Methods for screening ligands that activate the translocation of a steroid receptor to the nucleus in mammalian cells

ABSTRACT

The present invention provides a method of screening for a compound that binds to a selected nucleic acid comprising contacting compound fluorescently labeled by a fluorescent protein with a cell having a plurality of copies of the nucleic acid in an array such that the nucleic acid can be directly detected when bound by fluorescently labeled compound; and directly detecting the location of fluorescence within the cell, fluorescence aggregated at the site of the nucleic acid array indicating a compound that binds to the selected nucleic acid. In particular compounds such a transcription factors can be screened. Reagents for such method are provided including a mammalian cell having a plurality of steroid receptor response elements in an array such that the response element can be directly detected when bound by fluorescently labeled steroid receptor and a chimeric protein comprising a fluorescent protein fused to a steroid receptor.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of binding of molecules suchas transcription factors to regions of nucleic acids, steroid hormoneusage, steroid receptors and their corresponding response elements.Reagents are provided to allow methods involving direct detection ofbinding of a molecule, determining response element targeting byactivated steroid receptors, screening for steroid agonists andantagonists, and monitoring levels of steroid agonists and antagonistsin biological samples.

2. Background Art

Steroid receptors are hormone-dependent activators of gene expression.Steroid receptors mediate the action of steroid hormones (e.g.,glucocorticoids, estrogens, progestins, testosterone, mineralocorticoidsand 1,25-dihydroxycholecalciferol) in human tissues. After activationwith the cognate ligand, receptors bind to chromatin in the nucleus andmodulate the activity of target cellular genes. The binding of receptorsto these target sequences is a key step in steroid function. Currently,this interaction can only be detected by indirect methods, such asreporter assays that detect the result of transcriptional activationcoupled with transfection methods that introduce DNA sequences withreceptor binding sites.

It is generally accepted that the unliganded glucocorticoid receptor(GR) resides in the cytoplasm, and that hormone activation leads both tonuclear accumulation and gene activation. (Gasc, J. -M. & Baulieu, E. E.(1987) in Steroid Hormone Receptors: Their Intracellular Localisation,ed. Clark, C. R. (Ellis Horwood Ltd., Chichester, England), pp. 233-250;Beato, M. (1989) Cell 56, 335-344; Carson-Jurica, M. A., Schrader, W. T.& O'Malley, B. W. (1990) Endocr. Rev. 11, 201-220; Gronemeyer, H. (1993)in Steroid Hormone Action, ed. Parker, M. G. (Oxford University Press,New York), pp. 94-117; Tsai, M. J. & O'Malley, B. W. (1994) Annu. Rev.Biochem. 63, 451-486; Akner, G., Wikstrom, A. C. & Gustafsson, J. A.(1995) J. Steroid Biochem. Mol. Biol. 52, 1-16), and references therein.However, the mechanisms involved in nuclear translocation and targetingof steroid receptors to regulatory sites in chromatin have been poorlyunderstood. It has previously been difficult to discriminate between theability of a given receptor mutant, or a given receptor/ligandcombination, to participate in the separate processes of receptoractivation, nuclear translocation, sequence-specific binding, andpromoter activation.

Proteins have previously been labeled with fluorescent tags to detecttheir localization and sometimes their conformational changes both invitro and in intact cells. Such labeling is essential both forimmunofluorescence and for fluorescence analog cytochemistry, in whichthe biochemistry and trafficking of proteins are monitored aftermicroinjection into living cells (Wang, Y. L. & Taylor, D. L., eds.(1989) Methods Cell Biol. 29). Traditionally, fluorescence labeling isdone by purifying proteins and then covalently conjugating them toreactive derivatives of organic fluorophores. The stoichiometry andlocations of dye attachment are often difficult to control, and carefulrepurification of the proteins is usually necessary. If the proteins areto be used inside living cells, a final challenging step is to get themacross the plasma membrane via micropipet techniques or various methodsof reversible permeabilization. Furthermore, in previous hormone studiesbroken cell preparations or antibody tags in fixed cell preparationswere used, both techniques that cause enormous disruption of cellstructures.

The green fluorescent protein (GFP) from the jellyfish Aequorea Victoriais a molecule whose natural function seems to be to convert the bluechemiluminescence of the Ca²⁺-sensitive photoprotein aequorin into greenemission (Ward,W. W. (1979) in Photochemical and PhotobiologicalReviews, ed. Smith, K. C. (Plenum, N.Y.), 4:1-57). GFP's absorptionbands in the blue (maximally at a wave length of 395 nm with weakerabsorbance at 470 nm) and emission peak in the green (at 509 mn) do notarise from a distinct cofactor but rather from an internalp-hydroxybenzylideneimidazolidinone chromophore generated by cyclizationand oxidation of a serine-tyrosine-glycine sequence at residues 56-67(Cody, C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G. & Ward,W. W. (1993) Biochemistry 32, 1212-1218). The gene for GFP was cloned(Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. &Cornier, M. J. (1992) Gene 111, 229-233), and the encoded proteinconsists of 238 amino acid residues (molecular weight 27 kD).Heterologous expression of the gene has been done in Escherichia coli(Heim, R., Prasher, D. C. and Tsien, R. Y. (1994) Proc. Nati Acad. Sci.U.S.A. 91,12501-12504); Inouye, S. & Tsuji, F. I. (1994) FEBS Lett. 341,277-280), Caenorhabditis elegans (Chalfie, M., Tu, Y., Euskirchen, G.,Ward, W. W. & Prasher, D. C. (1994) Science 263, 802-805), andDrosophila melanogaster (Yeh, E., Gustafson, K. & Boulianne, G. L.(1995) Proc. Natl. Acad. Sci. U.S.A. 92, 7035-7040; Tannahill, D., Bray,S. & Harris, W. A. (1995) Dev. Biol. 168, 694-697 and plants (Hu,W. &Cheng, C. L. (1995) FEBS Lett. 369, 331-334; Baulcombe, D. C., Chapman,S. & Santa Cruz, S. (1995) Plant J. 7, 1045-1053). Recently, chimericgenes encoding N- and C-terminal fusions of the Drosophila exuperantia(exu) gene product, Exu (Wang, S. and Hazelrigg, T. (1994) Nature 369,400-403), actin Act88F gene (Barthmaier, P. and Fyrberg, E. (1995) Dev.Biol. 169, 770-774), and a nuclear localization signal (Davis, I.,Girdham, C. H. & O'Farrell, P. H. (1995) Dev. Biol. 170, 726-729); ofthe yeast microtubule and spindle pole associated dis1 gene product(Nabeshima, K., Kurooka, H., Takeuchi, M., Kinoshita, K., Nakaseko, Y.,& Yanagida, M. (1995) Genes Dev. 9, 1572-1585) and an RNA bindingprotein Npl3p (Corbett, A. H., Koepp, D. M., Schlenstedt, G., Lee, M. S.Hopper, A. K. & Silver, P. A. (1995) J. Cell Biol. 130, 1017-1026); andof a mammalian ion channel protein, NMDAR1 (Marshall, J., Molloy, R.,Moss, G. W., Howe, J. R. & Hughes, T. E. (1995) Neuron 14, 211-215),microtubule-associated protein, MAP4 (Olson, K. R., McIntosh, J. R. &Olmsted, J. B. (1995) J. Cell Biol. 130, 639-650), and a secretoryprotein, chromogranin B (Kaether, C. & Gerdes, H. H. (1995) FEBSLett.369, 267-271) have been constructed fused to GFP. However, none of thesechimeric proteins have been to transcription factors or co-factors andno suggestions have been made as to the usefulness of such a fusion tostudy physiologically relevant interaction on an amplified DNA target.Furthermore, none of these reports indicated a successful use of GFP inmammalian cells.

Many human diseases result from aberrant steroid function, and manydisease states, i.e., inflammation, are treated with glucocorticoid andother steroid derivatives. A large number of drugs have been developedwhose function is based on the ability to interact with and activatesteroid receptors. The identification and characterization of thesecompounds is a laborious, time-consuming and expensive process involvingyears of work. Even with a large investment of resources, the truebehavior of these compounds in living cells is not understood.

The present invention allows observation for the first time of in vivotarget sites within a higher eukaryotic nucleus for trans-regulatorymolecules, such as transcription factors, e.g., glucocorticoid receptor(GR). The visualization of physiologically relevant in vivo target sitesfor any transcription factor to date has not previously beenaccomplished. The present invention provides a poweful method foridentification of any single target site in a higher eukaryotic genome,comprising roughly 60,000-80,000 genes (Bird, A. P. (1995) Trends Genet.11:94-100), using a singly fluorescently-labelled regulatory factor,which has not been considered previously. Discriminating direct versusindirect interaction between a regulatory molecule and its putativeregulatory site is critical for the development of highly specific drugsdirected against trans-regulatory factors. Traditionally, themethodology for showing potentially direct interactions involvesnuclease or chemical protection experiments and transientco-transfection experiments of the putative regulator and its regulatedsite. While this approach indicates potential direct interaction, itdoes not necessarily imply direct interaction. Alternatively, theapproach of making compensatory mutations between the regulatorysequences as well as the DNA binding specificity has been used in anattempt to demonstrate direct regulatory interaction (Schier, A. F. aidGehring, W. J. (1992) Nature 356:804-807), an extension of theprinciples of second site suppression in genetics to molecular biology.However, such an approach makes enormous assumptions of ourunderstanding of sequence-specific recognition by sequence-specific DNAbinding proteins in vivo, which certainly would not be valid for manysystems, since many profound developmental events are governed byexquisite interactions to fine tune the system regarding, for example,concentration gradients of trans-regulatory factors. The presentinvention allows a simple and straight-forward manner in which directinteraction between a sequence-specific DNA binding protein or itsco-factor and its putative regulatory site in the in vivo genomiccontext can be addressed. With this simple inventive methodology, novelclasses of drugs directed not only against members of thesteroid-ligand-dependent transcription factors but to new classes ofdrugs that target other transcription factors or their co-factors can bescreened.

Additionally, the present invention provides the first opportunity toobserve and monitor gene targeting specifically of steroid receptors inliving cells wherein binding of the steroid receptor to its responseelement target can be observed distinctly from translocation of steroidreceptor. The invention therefore provides for many relevant analyses,such as real-time determination of steroid activity in subjects as wellas screening of compounds for response element binding/targetingcapabilities as distinct from translocation capabilities. Such methodshave implications in many diseases associated with steroid hormones,such as endocrine disorders, rheumatic disorders, collagen diseases,dermatological diseases, allergic states, ophthalmic diseases,respiratory disease, hematologic disorders, neoplastic disease,edematous states, gastrointestinal diseases and neurological conditions,and in other uses such as prevention of rejection of transplantedtissues.

SUMMARY OF THE INVENTION

The present invention provides a mammalian cell having a plurality ofsteroid receptor response elements in an array such that the responseelement can be directly detected when bound by fluorescently labeledsteroid receptor.

The present invention further provides a chimeric protein comprising afluorescent protein fused to a transcription factor. The presentinvention also provides a chimeric protein comprising a fluorescentprotein fused to a steroid receptor.

The instant invention provides an isolated nucleic acid encoding achimeric protein comprising a fluorescent protein fused to atranscription factor and an isolated nucleic acid encoding a chimericprotein comprising a fluorescent protein fused to a steroid receptor.

The instant invention also provides a cell containing a nucleic acidencoding a chimeric protein comprising a fluorescent protein fused to atranscription factor and a cell containing a nucleic acid encoding achimeric protein comprising a fluorescent protein fused to a steroidreceptor.

The instant invention provides a method of screening for a compound thatbinds to a selected nucleic acid comprising:

-   -   a. contacting compound fluorescently labeled by a fluorescent        protein with a cell having a plurality of copies of the nucleic        acid in an array such that the nucleic acid can be directly        detected when bound by fluorescently labeled compound; and    -   b. directly detecting the location of fluorescence within the        cell, fluorescence aggregated at the site of the nucleic acid        array indicating a compound that binds to the selected nucleic        acid.

The present invention also provides a method of characterizing aligand's effect on cellular localization of a compound to which theligand binds in a cell comprising:

-   -   a. contacting the ligand with a cell having the compound        fluorescently labeled by a fluorescent protein and    -   b. directly detecting the location of fluorescence within the        cell, the location of fluorescence in the cell indicating the        localization effect of the ligand on the compound.

Additionally provided is a method of determining a binding site for aDNA-binding protein comprising:

-   -   a. contacting the DNA-binding protein fluorescently labeled by a        fluorescent protein with a cell having a plurality of copies of        a nucleic acid having a putative binding site in an array such        that the putative binding site can be directly visualized when        bound by the fluorescently labeled DNA-binding protein, and    -   b. directly detecting the location of fluorescence within the        cell, the presence of fluorescence aggregated at the putative        binding site indicating a binding site to which the DNA-binding        protein binds.

The present invention also provides a method of screening for a ligandthat activates gene targeting of a steroid receptor in the nucleus of amammalian cell comprising:

-   -   a. contacting the ligand with a mammalian cell having a        plurality of steroid receptor response elements in an array such        that the response element can be directly detected when bound by        fluorescently labeled steroid receptor and the cell further        comprising a nucleic acid encoding a chimeric protein wherein a        fluorescent protein is fused to the steroid receptor; and    -   b. directly detecting the location of fluorescence within the        cell, fluorescence aggregated at the site of the steroid        receptor response element array in the nucleus indicating a        ligand that activates the gene targeting of a steroid receptor        in the nucleus of a mammalian cell.

The present invention provides a method of screening for a ligand thatactivates the translocation of a steroid receptor to the nucleus in amammalian cell comprising:

-   -   a. contacting the ligand with a mammalian cell having a        plurality of steroid receptor response elements in an array such        that the response element can be directly detected when bound by        fluorescently labeled steroid receptor and the cell further        comprising a nucleic acid encoding a chimeric protein wherein a        fluorescent protein is fused to the steroid receptor; and    -   b. directly detecting the location of fluorescence within the        cell, the location of fluorescence aggregated in the nucleus        indicating a ligand that activates the translocation of a        steroid receptor to the nucleus in a mammalian cell.

The instant invention provides a method of detecting in a biologicalsample the presence of an agonist of a steroid receptor comprising:

-   -   a. contacting the sample with a mammalian cell having a        plurality of steroid receptor response elements in an array such        that the response element can be directly detected when bound by        fluorescently labeled steroid receptor and the cell further        comprising a nucleic acid encoding a chimeric protein wherein a        fluorescent protein is fused to the steroid receptor; and    -   b. directly detecting the location of fluorescence within the        cell, the location of fluorescence aggregated at the site of the        steroid receptor response element array in the nucleus        indicating the presence of an agonist of the steroid receptor in        the sample.

The present invention also provides a method of detecting in abiological sample the presence of an antagonist of a steroid receptorcomprising:

-   -   a. contacting the sample and an agonist of the steroid receptor        with a mammalian cell having a plurality of steroid receptor        response elements in an array such that the response element can        be directly detected when bound by fluorescently labeled steroid        receptor and the cell further comprising a nucleic acid encoding        a chimeric protein wherein a fluorescent protein is fused to the        steroid receptor; and    -   b. directly detecting the location of fluorescence within the        cell, the absence of fluorescence substantially aggregated at        the site of the steroid receptor response element array in the        nucleus indicating the presence of an antagonist of the steroid        receptor in the sample.

The present invention provides a method of monitoring the level of anagonist of a steroid receptor in a subject comprising:

-   -   a. periodically obtaining a biological sample from the subject,    -   b. contacting the sample with a mammalian cell having a        plurality of steroid receptor response elements in an array such        that the response element can be directly detected when bound by        fluorescently labeled steroid receptor and the cell further        comprising a nucleic acid encoding a chimeric protein wherein a        fluorescent protein is fused to the steroid receptor; and    -   c. directly detecting the location of fluorescence within the        cell, a decrease in fluorescence aggregated at the site of the        steroid receptor response element in the nucleus in a        later-obtained sample relative to an earlier-obtained sample        indicating a decrease in level of the steroid agonist of the        steroid receptor in the sample and an increase in fluorescence        aggregated at the site of the steroid receptor response element        in the nucleus in a later-obtained sample relative to an        earlier-obtained sample indicating an increase in level of the        steroid agonist of the steroid receptor in the sample.

The instant invention provides a method of monitoring the balancebetween levels of an agonist of a steroid receptor and an antagonist ofthe steroid receptor in a subject comprising:

-   -   a. periodically obtaining a biological sample from the subject,    -   b. contacting the sample with a mammalian cell having a        plurality of steroid receptor response elements in an array such        that the response element can be directly detected when bound by        fluorescently labeled steroid receptor and the cell further        comprising a nucleic acid encoding a chimeric protein wherein a        fluorescent protein is fused to the steroid receptor; and    -   c. directly detecting the location of fluorescence within the        cell, an increase in fluorescence aggregated at the site of the        steroid receptor response element in the nucleus in a        later-obtained sample relative to an earlier-obtained sample        indicating an increase in level of the steroid agonist relative        to level of the steroid antagonist in the sample, and a decrease        in fluorescence aggregated at the site of the steroid receptor        response element in the nucleus in a later-obtained sample        relative to an earlier-obtained sample indicating an increase in        level of the steroid antagonist of the steroid receptor relative        to level of the steroid agonist in the sample.

The instant invention also provides a method of determining an effectivedosage of a steroid receptor agonist in a subject comprising:

-   -   a. transferring into a set of cells from the patient a nucleic        acid encoding a chimeric protein comprising a fluorescent        protein fused to a steroid receptor;    -   b. contacting the cells in the set with one of a selected range        of dosages of the steroid agonist; and    -   c. directly detecting location of fluorescence in the set of        cells, a dosage capable of locating fluorescence substantially        in the nucleus indicating an effective dosage of steroid        receptor agonist.

The present invention provides a method of determining an effectivedosage of a steroid receptor agonist to maintain steroid receptoractivation for a selected period of time in a subject comprising:

-   -   a. administering to the subject a dosage of the steroid receptor        agonist,    -   b. periodically obtaining a biological sample from the subject,    -   c. contacting the sample with a mammalian cell having a        plurality of steroid receptor response elements in an array such        that the response element can be directly detected when bound by        fluorescently labeled steroid receptor and the cell further        comprising a nucleic acid encoding a chimeric protein wherein a        fluorescent protein is fused to the steroid receptor; and    -   d. directly detecting the location of fluorescence within the        cell, a dosage that maintains the location of fluorescence at        the site of the steroid receptor response element array in the        nucleus for the selected period of time indicating an effective        dosage.

The present invention also provides a method of determining an effectivedosage of a steroid receptor antagonist to abrogate agonist activity fora selected period of time in a subject comprising:

-   -   a. administering to the subject a dosage of the steroid receptor        agonist,    -   b. periodically obtaining a biological sample from the subject;    -   c. contacting the sample with a mammalian cell having a        plurality of steroid receptor response elements in an array such        that the response element can be directly detected when bound by        fluorescently labeled steroid receptor and the cell further        comprising a nucleic acid encoding a chimeric protein wherein a        fluorescent protein is fused to the steroid receptor; and    -   d. directly detecting the location of fluorescence within the        cell, a dosage that prevents the location of fluorescence at the        site of the steroid receptor response element array in the        nucleus for the selected period of time indicating an effective        dosage.

The present invention also provides a method of detecting a defect in aresponse pathway of a steroid receptor in a subject comprisingtransferring into a cell from the subject a nucleic acid functionallyencoding a chimeric protein comprising a fluorescent protein fused tothe steroid receptor and detecting the location of fluorescence withinthe cell as compared to the location of fluorescence within a normal,control cell transfected with the nucleic acid, a difference in locationof fluorescence within the cell of the subject as compared to locationof fluorescence within the normal, control cell indicating a defect inthe response pathway of the steroid receptor.

The instant invention provides a method of determining whether a defectin a response pathway of a steroid receptor in a subject is intranslocation of the steroid receptor to a cell nucleus, comprisingtransferring into a cell from the subject having the defect a nucleicacid functionally encoding a chimeric protein comprising a fluorescentprotein fused to the steroid receptor and detecting the location offluorescence within the cell, the location of fluorescence substantiallyin the cytoplasm of the cell indicating the defect is in translocationof the steroid receptor to the nucleus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the integrated, tandem array of bovine papillomavirus (BPV) 69% transforming fragment, mouse mammary tumor virus (MMTV)long terminal repeat (LTR) containing glucocorticoid receptor (GR)binding sites, and Harvey murine sarcoma virus v-ras sequences(Ha-v-ras).

FIG. 2 shows, in panels 11-17, the appearance of GFP-GR when thefluorescent tag is excited by 489 nm laser light, and the 511 nmemission examined by confocal microscopy, using a standard fluoresceinfilter set. Individual sections are depicted for a representative 3134cell nucleus. As one focuses on 0.18 micrometer sections throughout thenucleus, a continuous fiber of intense light emission is detected over5-7 sections, which corresponds to GFP-GR binding to the continuousBVP/MMTV-LTR/ras array. The array is seen in sections 12-16. Below thepanels is provided a schematic presentation of the appearance of GFP-GRwhen the fluorescent protein is excited.

FIG. 3 shows the construction of GFP-GR. (A) Plasmid pCI-nGFP-C656Gcontains the green fluorescent protein fused to the C656G mutantglucocorticoid receptor. (B) Dexamethasone (dex) dependent stimulationof MMTV-pLTRLuc is shown for GFP-GR transfected cells. Solid barsrepresent the IL2R⁺ selected population activated with 1 nM dex, and theopen bar depicts activation of the endogenous receptor with 100 nM dex.(C) Ligand specificity is presented for activation of endogenousMMTV-LTR-CAT sequences present in the 1471.1 cells. Cells were treatedfor 4 hrs with the indicated ligand, then harvested and levels of CATactivity determined.

FIG. 4 shows nuclear localization of GFP-GR in MCF7 cells when the cellsare treated with (A) dexamethasone or (B) RU486. The fluorescent tag isexcited by 489 nm laser light, and the 511 nm emission examined byconfocal microscopy, using a standard fluorescein filter set.

FIG. 5 shows nuclear localizationof GFP-ER in (A) MCF7 cells and (B)MDA-MB-231 cells when the fluorescent tag is excited by 489 nm laserlight, and the 511 nm emission examined by confocal microscopy, using astandard fluorescein filter set. MCF7 cells are reported to be estrogenreceptor positive and hormone dependent. MDA-MB0231 cells are reportedto be estrogen receptor negative and hormone independent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides cell lines such as the murine cell line3134, that contains a set of mouse mammary tumor virus (MMTV) Harveymurine sarcoma virus (HaMuSV) v-ras sequences organized in ahead-to-tail tandem array of approximately 200 copies. Each MMTVpromoter sequence in this array contains 4 glucocorticoid receptor (GR)binding sites; the complete array thus contains nearly 1000 GR bindingsites. This cell is used to visualize directly the interaction betweenGR and its binding site in chromatin in living cells. This isaccomplished with a fluorescent labeled copy of the GR. This array thusprovides the unique opportunity to visualize direct receptor/targetinteractions. This structure, for which there is no precedent, providesfor the first time a reagent to observe the interaction of steroidreceptors with their response elements in living cells, and tocharacterize the effectiveness of medically important steroid ligands inactivating gene expression in mammalian cells.

In the present application, we have shown the usefulness of GFP inmonitoring the activity of a steroid hormone receptor. The ability todirectly observe living cells has allowed us to follow in real time theprocess of cytoplasm to nuclear translocation, and has revealed for thefirst time differences in GR intranuclear accumulation pattern dependenton the type of activating ligand. Furthermore, the patterns of GRaccumulation are remarkably similar between adjacent cells, suggestingan order in the organization of the interphase nucleus. This ability toobserve living cells has also revealed subcellular localization ofpartially activated estrogen receptor (ER). Thus, the use of GFP hasrevealed new details about steroid localization and organization of theeukaryotic nucleus.

As used in the specification and in the claims, “a” can mean one ormore, depending upon the context in which it is used.

The present invention provides reagents and methods for detecting, bydirect visual evaluation, the binding of fluorescently labelledcompounds to a nucleic acid. This direct detection can be accomplishedby the use of a cell line having a sufficient number of copies of thebinding region of the nucleic acid in an array, such as tandem repeats,that allows detection of the array when a fluorescently labeled compoundis bound to the binding region by direct detection of the fluorescencelocalized at the site in the cell nucleus of the nucleic acid array. Forexample, after binding, cells can immediately, without further treatmentof the cells, be placed under a fluorescent microscope and fluorescencedirectly visualized. Thus compounds can be rapidly analyzed for bindingcapability in a manner that clearly depicts the binding. Importantly,the binding detected in this assay is biologically relevant. Thedetected event represents receptor mobilization to a correct genetictarget in the living cell. Thus, interactions revealed with this assayare much more reliable as measures of biologically germane receptoractivity. Additionally, analyses of levels of compounds or defects inpathways involving the binding of such compounds to their nucleic acidbinding site in specific subjects can be performed, as further describedbelow.

Any selected nucleic acids and binding sites, and compounds that maybind thereto, either directly or indirectly, can be analyzed by thismethod, in a desired cell, as elaborated below. For example, binding ofany transcription factor to its activation site on a nucleic acid can bedirectly determined along with the proteins associated with the factor(e.g., co-activator, co-repressor, adapter, or molecules in a similarcategory). Additionally, for example, binding of any selected steroidreceptor can be directly detected and analyzed for both translocation tothe nucleus and binding to the corresponding response element(s) in thenucleus. Thus, compounds typically can be proteins, polypeptides andpeptides; however, other compounds can include, for example, PeptideNucleic Acids (PNAs), antisense nucleic acids and organic molecules(e.g., dexamethasone). Importantly, for any compound, the step ofbinding to the binding site can visually be distinguished from the stepof translocation to the nucleus, or alternatively, exit from thenucleus.

In general, the present invention utilizes fluorescent labeling of thecompound by a fluorescent protein, as fluorescent protein is hereindescribed, adding the labeled compound to cells, and directly detectingthe location and/or aggregation of fluorescence in the cells. Fordetection of the translocation of the labeled compound to the nucleus,any cell can be utilized, since the resulting location of fluorescencecan be visualized as either in the cytoplasm or in the nucleus.Additionally, for such detection events, cells having increased copynumber of the binding site, in any array, can be used. For detection ofbinding to the target nucleic acid site, the present invention providescells having a plurality of nucleic acid binding sites in an array suchthat the nucleic acid binding site can be directly detected when boundby binding compound, such as a ligand, transcription factor, etc.,fluorescently labeled by a fluorescent protein, as described herein.

As used herein, direct detection means detection of the fluorescenceemitted from the site in the cells when excited by light, ultraviolet orvisible, without the need for any additional chemical reactions ortreatment of the cells. The fluorescence is directly detected by anydevice capable of detecting fluorescence, such as a fluorescentmicroscope, as visualized by the eye of the operator of the microscopeat the time or as recorded from the microscope such as by photography ofthe field of view or through the use of photosensitive detectors. Afluorescent microscope, such as a confocal laser scanning microscope oran epifluorescent microscope, can be used, as is known in the art. Thereis no requirement that cells be, e.g., fixed or stained or contacted byany additional reagents, in order to detect the binding. Thus livingcells can be assayed, and results obtained, immediately after binding.Therefore, for example, subjects can be advised immediately of resultsof analyses as described below. Furthermore, it is anticipated thatscreening of both nuclear localization of fluorescence (translocation)and focal localization of fluorescence on a target array will be adaptedto high volume computerized image analysis. That is, the analysis oflarge numbers of samples will be automated for either the repetitiveexamination of clinical samples or the large-scale screening ofcompounds in the research environment.

The cell can be derived from any desired mammal, such as, for example,human, monkey, mouse, hamster and rat. The nucleic acid can be amplifiedin an appropriate array by any of several means, as known to thoseskilled in the art. Generally, a selected nucleic acid binding site orcollection of sites, for example as found within the context of atranscriptional regulatory region, i.e., promoters, enhancers,silencers, etc., can be amplified in an array detectable by the presentmeans, for example, by gene amplification of the nucleic acid bindingsite (e.g., the steroid receptor response element or the transcriptionfactor binding site) by linking it to a gene readily amplified in atandem array, for example, dihydrofolate reductase, or bymultimerization of the nucleic acid binding site or sites by syntheticDNA synthesis and/or enzymatic synthesis, for example, through the useof ligase and polymerases, and introducing the amplified element intoselected mammalian cells. Such methods are further elaborated in theexamples provided below.

The cells used herein have an array of the nucleic acid having bindingsites under analysis such that the nucleic acid can be directly detectedwhen bound by a fluorescently labeled compound. Such array ascontemplated herein has sufficient copies of the nucleic acid and insuch an arrangement that the fluorescently labeled binding compound,when bound to the site can be directly detected and readily identified.Therefore, the array includes arrangement of the copies in sufficientlyclose physical proximity along a chromosome, either present endogenouslyor artificially introduced or induced, or in extrachromosomallyreplicating episomes, to allow localization of fluorescence at adiscrete, detectable site in the nucleus, as seen under standardmagnification for cells and nuclei. Such array as contemplated hereinallows detection in the context of chromatin, as exists in theinterphase nucleus. An example of such an array is a series of directtandem repeats of the nucleic acid. An example of a tandem array ofdirect repeating units is depicted in FIG. 1.

By a “plurality” of any herein described nucleic acid having a bindingsite is meant that the number of copies of the nucleic acid having thebinding site (e.g., the steroid receptor response element or thetranscription factor binding site) is greater than one. Preferably, thecells have more than about five copies, more preferably more than aboutten copies, more preferably more than about twenty, and more preferablymore than about forty copies. For example, cell line 3134, describedherein, has about two hundred copies of the MMTV LTR-Ha-v-ras-gene, eachof which has four copies of the binding site for glucocorticoid receptorand with each site accommodating two glucocorticoid receptor molecules.Any number which allows detection of the site upon binding of thefluorescently labeled binding compound is contemplated. Thus, an exampleof a cell of the present invention is a cell of the cell line 3134deposited with American Type Culture Collection 10801 University Blvd.,Manassas. VA 20110-2209, under the provisions of the Budapest Treaty onDecember 11, 1995 and has been accorded ATCC accession number: CRL-11998(ATTC).

Specifically, the present invention provides a cell having a pluralityof steroid receptor response elements in an array such that the responseelement can be directly detected when bound by fluorescently labeledsteroid receptor. A response element, as used herein, includes anynucleic acid to which a steroid receptor directly binds, but alsoincludes the steroid receptor associated, either directly or indirectly,factors that are recruited to the vicinity of the element (e.g., nuclearfactor 1 (NF1), octomer transcription factor 1 (OTF1), steroid receptorcoactivator 1 (SRC1), etc.). Steroid receptors, and correspondingresponse elements to which they bind, can include any steroid receptor,for example, glucocorticoid receptor, estrogen receptor (ER),progesterone receptor, androgen receptor, mineralocorticoid receptor,vitamin D receptor. Examples of steroid receptor response elementsinclude those contained in the mouse mammary tumor virus (MMTV) longterminal repeat (LTR) (which has binding sites for glucocorticoidreceptor, mineralocorticoid receptor, progesterone receptor, andandrogen receptor), and those contained in vitellogenin and osteocalcingenes (which have binding sites for estrogen and vitamin D receptors).Thus, response elements in such array in a cell can include othertranscriptional regulatory elements contained within the mouse mammarytumor virus long terminal repeat and bovine pappilloma virus 69%transforming DNA. Many steroid receptors and steroid response elements,as exemplified above, are known to the skilled artisan; however, anysteroid receptor and its response element is contemplated herein. Inaddition to steroid receptors, there are other ligand-dependentreceptors (such as thyroid hormone receptor, retinoic acid receptor,retinoid X receptor, TCCD (dioxin) receptor, fatty acid activatablereceptors, etc.) and stimulus-dependent receptors (such as peroxisomeproliferator activated receptor, growth factor-dependent receptors(e.g., epidermal growth factor, nerve growth factor, etc.)), and factors(such as CREB, NFAT, NFkB/IkB, etc.), and other receptors whose ligandremains to be defined (such as mammalian homologs of the Drosophilatailless, knirps, sevenup, FTZF1 genes, etc.). Many of these receptorsor factors can be found listed in the book [Parker, M. G. (1993) SteroidHormone Action (Oxford University Press, New York, pp. 210)], in arecent review article [Tsai, M. J. & O'Malley, B. W. (1994) Annu. Rev.Biochem. 63, 451-486], and in the GenBank database, which will containadditional receptors as well as the complete nucleotide sequences of thegenes and cDNAs. In addition, the cell line offers a number of tandemlyrepeated regulatory sites for sequence-specific transcription factors(such as activating protein 2 (AP2), OTF1, NF1/CTF, etc.) as well asgeneral transcription factors (such as TFIID, initiator protein, etc.).The steroid receptor response elements (or other transcriptionalregulatory elements) used in the present invention in arrays detectableas described herein can be integrated into the genome of the cell,maintained in the cell on artificial mammalian chromosomes (Monaco, A.P. and Larin, Z. (1994) Trends Biotechnol. 12, 280-286) or can becarried on episomal elements.

The cell can further comprise a nucleic acid encoding a chimeric proteinwherein a fluorescent protein is fused to the steroid receptor. Steroidreceptors as described above are contemplated for use in a chimericprotein. Typically, the steroid receptor in the chimeric protein encodedby the nucleic acid in a selected cell is one that binds to the responseelement in the selected cell in the herein described array. Throughoutthis application by “a fluorescent protein” is meant a protein thatfluoresces in cells without adding exogenous cofactors. That is, it is aprotein that can be expressed in cells and detected in these cellssimply by exciting the protein with light and visualizing the resultantfluorescence. An example of such a fluorescent protein is the greenfluorescent protein (GFP) originally isolated from the jellyfishAequorea victoria. Another example of a fluorescent protein as definedherein is the green fluorescent protein originally isolated from Renillareniforms, which demonstrated a single absorption peak at 498 nm and anemission peak at 509 nm. (Cubitt, et el. (1995) TIBS 20: 448-455). Byfluorescent protein is also contemplated that modifications may be madeto a fluorescent protein, as long as the resulting protein fluoresceswhen expressed in cells. Modifications can be developed based upon thechemistry of chromophore formation. (Cubitt, et al. TIBS 20: 448-455).Generally, however, one may prefer to leave the glycine whichparticipates in forming the chromophore, in part by cyclization of aSer-Tyr-Gly moiety (Gly 67 in Aequorea GFP in the Ser65-Tyr66-Gly67moiety), intact. An example of a useful substitution that modifies theabsorption spectra is the substitution in Aequorea green fluorescentprotein for serine at amino acid 65 by, for example, threonine,cysteine, leucine, valine, or alanine, that allows the excitation of thechromophore at a lower energy (longer wavelength) than the naturallyoccurring protein thereby greatly decreasing the destruction of thechromophore as occurs when it is excited at a higher energy. Suchmutation at amino acid 65 also increases brightness and rate ofoxidation as compared to wild-type Aequorea GFP when each is excited atits longest wavelength peak. In addition, other spectral variants ofGFP, such as improved blue variants of GFP have been developed (e.g.,pCI-nGL2-C656G; pCI-nGL3-C656G; pCI-nGL4-C656G; pCI-nGL5-C656G;pCI-nGL7-C656G; pCI-nGL9-C656G; pCI-nGL10-C656G; pCI-nGL11-C656G).Variants emitting longer wavelengths (e.g., red variants) can also bedeveloped by introducing other mutations into the GFP DNA. Additionally,the codon usage of any GFP-coding sequence can be modified to humancodons, according to known methods. For example, pGreenLantern-1(LifeTechnologies, Inc., Gaithersburg, Md., catalog number 10642-015)isa commercially available S65T variant GFP cDNA with mammalian codonusage. Other commerically available humanized GFP-cDNAs are: pEGFP-N(catalog numbers: 6086-1; 6085-1; 6081-1 from Clontech), pEGFP-C(catalog numbers: 6084-1; 6083-1; 6082-1 from Clontech), and pHGFP-S65T(catalog number 6088-1 from Clontech). Additional useful modificationsof any fluorescent protein can include other modifications that speed upthe rate of the oxidation step of chromophore formation, that increasebrightness at longer wavelengths, and that reduce pohotoisomerizationand/or photobleaching. Furthermore, in general it is preferable that GFPnot be truncated by more than about one amino acid from the aminoterminus and about 10-15 amino acids from the carboxyl terminus.Detection of additional fluorescent proteins can readily be performed bystandard approaches such as searching for proteins having some homologyto GFP in nucleic acid libraries from organisms that demonstratefluorescence by nucleic acid hybridization and by searching forhomologous nucleic acids and proteins in other organisms in databanks ofnucleic acid and protein sequences and testing the encoded proteins forfluorescence. It is possible that forced protein evolution of thecurrently existing GFP can be achieved by randomizing the entire GFPcoding region so as to make every single possible change at every singleamino acid coding region as well as pairs and further combination ofchanges. Desirable changes yielding better chromophore or differentexcitation/emission spectra can be characterized by fluorescencespectroscopy or flow cytometry upon translation of the coding sequencesinto proteins. An example of one general approach would be to takeadvantage of the phage display system for expression of the chromophoreon the surface of a bacteriophage using a modification of what iscurrently being done for antibodies (e.g. Pharmacia Biotech, Inc.'sRecombinant Phage Antibody System). Another general approach would be toadapt a protocol similar to that used to select novel enzymaticactivities displayed by RNAs (Bartel, D. P. and Szostak, J. W. (1993)Science 261, 1411-1418) for selection of novel fluorescent proteins.

Therefore, also provided herein are chimeric proteins comprising afluorescent protein fused to a transcription factor, and nucleic acidsencoding such proteins. One example of a transcription factor that canbe used herein, described in further detail above, is a steroidreceptor; however, numerous other transcription factors can be utilized.For example, basal transcription factors (e.g. TFIID, etc.), andsequence specific DNA binding transcription factors (e.g., AP1, AP2,SP1, NF1, etc). Additional transcription factors are listed in, forexample, computer databases such as that maintained by the NationalCenter for Biotechnology Information (NCBI, Bethesda, Md.) accessiblethrough the BLAST program (see item 19 (TFD) for transcription factors;item 20 for eukaryotic promoter sequences). Additionally, as used in theclaims, “transcription factors” include transcription adaptor moleculesor cofactors, which localization within the cell can be monitored alsoby this method. Transcription adaptor molecules or cofactors are thosemolecules that interact with transcription factors to effect theirfunction (i.e., their activation or repression functions). For example,SRC 1, steroid receptor coactivator 1, is a cofactor of steroidreceptors.

The chimeric protein can include a linking peptide sequence between thefluorescent protein and the steroid receptor. For example, a sequence ofthe amino acids glycine and alanine, or a sequence of alanine alone canbe used; however, any sequence of amino acids and any length can be usedthat does not interfere with the binding of the steroid receptor to itsresponse element and that does not prevent fluorescence of thefluorescent protein. Typically, a linker peptide will range from two toabout ten amino acids but maybe shorter or longer. Of course, certainlinker peptides maybe preferred over others, e.g., the presence of fourbasic amino acids in a string of six might suffice as a nuclearlocalization signal so as to mislocate the uninduced state of thefactor. A linker peptide can be used to separate the fluorescent proteinstructurally from the response element and can function to allow thefluorescent protein independently of the remaining portion of thechimeric protein. An example of a chimeric protein, which has GFP fusedvia five glycine-alanine repeating units to the N-terminus of theglucocorticoid receptor, is provided herein as SEQ ID NO: 2. Typically,a fluorescent protein can be fused to either the C-terminus or theN-terminus of the transcription factor; however, the preferableconstruction for a specific transcription factor can readily bedetermined. Linker peptides can readily be introduced between the twoproteins in the chimeric protein by producing an nucleic acid thatencodes the chimeric protein having the linker sequence between thefluorescent protein and transcription factor components.

Modifications to the fluorescent protein portion of the chimeric proteinand/or the transcription factor, e.g., steroid receptor can be made. Forexample, a green fluorescent protein can be modified as described above.The transcription factor, for example, can be modified to increase ordecrease its affinity for its binding site or to determine if a selectedmodification affects its binding affinity. In the case of steroid- orligand-dependent transcription factor, the region involved in steroid-or ligand-binding can be altered to either increase or decrease theaffinity to the steroid or ligand or alter the specificity of theligand. Furthermore, other functions of the factor, such astransactivation potential, maybe modified. An example of such amodification is found in the chimeric protein having the amino acidsequence set forth in SEQ ID NO: 2, wherein the transcription factor isglucocorticoid receptor having a substitution of serine for cysteine atamino acid 656 that has a higher binding affinity for its ligand thanthe protein having the naturally occurring amino acid sequence. Thissubstitution also increases the transactivation potential of thereceptor, resulting in “superactivation.”This cysteine 656 mutation canbe utilized, for example in rat, human and mouse glucocorticoidreceptor. For example, steroid receptors, or any transcription factor,can be modified in their steroid binding domains to increase affinityfor steroid, thus allowing one to increase use of exogenous receptorover endogenous receptor in a cell.

The transcription factor of the chimeric protein can be derived from anyselected mammal. Additionally, chimeric proteins utilizing atranscription factor from one mammal can often be used in a cell fromanother mammal. For example, the glucocorticoid receptor amino acidsequence is highly conserved, particularly in the binding region amongrat, human and mouse, and, for example, the rat glucocorticoid receptorbinds with high affinity to the human glucocorticoid response element.

Also provided in the present invention is nucleic acid encoding achimeric protein wherein a fluorescent protein is fused to atranscription factor. The nucleic acid encoding the chimeric protein canbe any nucleic acid that functionally encodes the chimeric protein. Forexample, to functionally encode, i.e., allow the nucleic acid to beexpressed, the nucleic acid can include, for example, expression controlsequences, such as an origin of replication, a promoter, an enhancer,and necessary information processing sites, such as ribosome bindingsites, RNA splice sites, polyadenylation sites, and transcriptionalterminator sequences. Preferred expression control sequences arepromoters derived from metallothionine genes, actin genes,immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus,etc. A nucleic acid encoding a selected chimeric protein can readily bedetermined based upon the genetic code for the amino acid sequence ofthe selected chimeric protein, and, clearly, many nucleic acids willencode any selected chimeric protein. Modifications to the nucleic acidsof the invention are also contemplated, since mutations in the steroidreceptor binding can thereby be studied for binding affinity.Additionally, modifications that can be useful are modifications to thesequences controlling expression of the chimeric protein to makeproduction of the chimeric protein inducible or repressible uponaddition to the cells of the appropriate inducer or repressor. Suchmeans are standard in the art (see, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989). The nucleic acids can be generated bymeans standard in the art, such as by recombinant nucleic acidtechniques, as exemplified in the examples herein, and by syntheticnucleic acid synthesis or in vitro enzymatic synthesis.

An example of a nucleic acid of the present invention is a nucleic acidencoding a chimeric protein comprising a green fluorescent protein fusedvia ten amino acid gly-ala linker to the N-terminus of the ratglucocorticoid receptor. One nucleic acid encoding this nucleic acid isset forth in SEQ ID NO: 1. This nucleic acid encodes a modified AequoreaVictoria green fluorescent protein and a modified rat glucocorticoidreceptor. Another example is a nucleic acid encoding a chimeric proteincomprising a modified Aequorea Victoria green fluorescent protein fused(via ten amino acid gly-ala linker) to the N-terminus of the humanestrogen receptor.

Additionally contemplated by the invention are closely related receptorsand nucleic acids encoding them. Thus, provided by the invention arenucleic acids that specifically hybridize to the nucleic acids encodingthe chimeric proteins under sufficient stringency conditions toselectively hybridize to the target nucleic acid. Thus, nucleic acidsfor use, for example, as primers and probes to detect or amplify thetarget nucleic acids are contemplated herein. Typically, the stringencyof hybridization to achieve selective hybridization is about 5° C. to20° C. below the Tm (the melting temperature at which half of themolecules dissociate from its partner). Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The washingtemperatures can similarly be used to achieve selective stringency, asis known in the art. (Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987).

The present invention provides cells containing a nucleic acid of theinvention. A cell containing a nucleic acid encoding a chimeric proteintypically can replicate the DNA and, further, typically can express theencoded protein. The cell can be a prokaryotic cell, particularly forthe purpose of producing quantities of the nucleic acid, or a eukaryoticcell, particularly a mammalian cell. The cell is preferably a mammaliancell for the purpose of expressing the encoded protein so that theresultant produced protein has mammalian protein processingmodifications. Additionally, as described above, the cell can have anarray of a nucleic acid to which the encoded chimeric protein binds.

Labeled compounds and nucleic acids encoding chimeric proteins can bedelivered into cells by any selected means, in particular depending uponthe purpose of the delivery of the compound and the target cells. Manydelivery means are well-known in the art. For example, electroporation,calcium phosphate precipitation, microinjection, cationic or anionicliposomes, and liposomes in combination with a nuclear localizationsignal peptide for delivery to the nucleus can be utilized, as is knownin the art. In particular for transfer of a nucleic acid into a cell, toenhance transfer a cotransfection of the nucleic acid with a secondnucleic acid encoding a selectable marker can be performed, andtransfected cells selected for by the selectable marker. For example,the interleukin 2 receptor (IL2R) gene can be cotransfected, andselection performed by using beads having the antibody to IL2R bound tothe beads to separate out transfected cells. Such methods are standardin the art.

Nucleic acids of the present invention can be used to generatetransgenic animals in which the nucleic acid encoding a selectedchimeric protein, such as GFP-GR for GR studies or GFP-ER for ERstudies, is added to the germ line of the animals. Thus a cell of theinvention containing an nucleic acid of this invention is contemplatedto include a cell in a transgenic animal. With such transgenic animals,cytoplasm-to-nucleus translocation and gene targeting can be observed inany tissue of interest. Thus studies over the life cycle of the animalcan be conducted, so that, for example, development and effects ofenvironment, aging, cancer, etc. can be readily observed. Transgenicanimals are generated by standard means known to those skilled in theart.

The provision of the present method to visualize physiologicallyrelevant target sites within the eukaryotic nucleus allows one todirectly observe nuclear target sites for any desired steroid- orligand-dependent transcription factors as well as any nuclearly targetedtrans-regulatory factors. Conceptually, the requirements are: 1) to tagthe protein of interest with a fluorescent protein, such as the greenfluorescent protein, using standard recombinant DNA techniques whereinthe two cDNAs are fused in frame to each other such that introductionback into the mammalian cell would give rise to the synthesis of thechimeric protein of interest; and, 2) to create an identifiable nucleartarget site by linking together multiple copies of the potential targetsite to generate a large enough array as to be readily discernible whenthe fluorescently labeled chimeric protein interacts with the targetsite. Such an interaction is visible as an intense, concentratedfluorescent signal unique to the cell harboring the array and absentfrom the parental cell lacking the array. Agents which modify theinteraction of the labeled factor with its cognate site can thus bereadily screened.

The present invention further provides a method of screening for acompound that binds to a selected nucleic acid comprising:

-   -   a. contacting compound fluorescently labeled by a fluorescent        protein with a cell having a plurality of copies of the nucleic        acid in an array such that the nucleic acid can be directly        detected when bound by fluorescently labeled compound; and    -   b. directly detecting the location of fluorescence within the        cell,        fluorescence aggregated at the site of the nucleic acid array        indicating a compound that binds to the selected nucleic acid.        Thus, the present method can be utilized to directly determine        whether a compound binds a nucleic acid, directly or indirectly.        For example, transcription factors that have been indirectly        shown to affect binding can now be assessed to determine if they        do bind the target DNA. If, by this method a factor labeled by a        fluorescent protein, as herein described (for example, Aequorea        GFP), is added to cells, and upon visualization of the        fluorescence, the fluorescence is seen to be aggregated at the        site of the target array of nucleic acid, then the factor, or a        factor(s) to which the labeled factor binds, binds the nucleic        acid. The nucleic acids are conceptualized as merely serving as        an easily identifiable “stage” upon which the “actors” (i.e.,        transcription factors and associated factors) appear.

In the present inventive methods, by fluorescence aggregated at the siteof the nucleic acid array is meant fluorescence aggregated at a singlepredominant site within the nucleus. Such aggregation can readily bedetected upon excitation of the fluorescent protein. Detection, asdescribed above, can be performed with the use of a fluorescentmicroscope. Magnification can be altered as desired for more or lessdetail in visualizing the aggregation, such as from about 60× to about200×, with a typical magnification being about 100×. The fluorescentprotein in any herein described method can be, for example, a greenfluorescent protein, as described herein.

Compounds for use in the present methods can be labeled by standardmeans in the art for linking a peptide to the compound. For example,when the compound to be labeled is a peptide, polypeptide or protein, achimeric protein can be made by synthesizing a nucleic acid that encodesthe chimeric protein having the fluorescent protein fused to thecompound protein. Proteins can also be labeled with a fluorescentprotein by a chemical bridge. Additionally, a fluorescent protein labelcan be placed on the compounds by non-covalent interaction, such as thatdisplayed by steroids with their binding domain [e.g., fluorescein orrhodamine conjugated dexamethasone (available through Molecular Probes,Inc. catalog number D-1382 or D-1383) for the glucocorticoid receptor].

More than one chimeric protein, preferably each comprising a fluorescentprotein that emits a different wavelength of light (for example amodification of Aequorea GFP that emits blue and a modification ofAequorea GFP that emits green), can be used simultaneously in thepresent inventive methods. For example, plasmids pCI-nGL2-C656G;pCI-nGL3-C656G; pCI-nGL4-C656G; pCI-nGL5-C656G; pCI-nGL7-C656G;pCI-nGL9-C656G; pCI-nGL10-C656G; pCI-nGL11-C656G, all GFP-GR having bluevariants of GFP (BFP) in a humanized codon usage (improved bluevariants) can be used with any GFP-GR having green GFP (such aspCI-nGFP-C656G, pCI-nGL1-C656G, pTET-nGFP-C656G and pOP-nGFP-C656G,exemplified herein) in this method.

Alternatively, two different fluorescent moieties that show distinctexcitation maxima with identical emission wavelengths could also besimultaneously utilized to detect the presence of both fluorescentlylabeled compounds on the same DNA array. With the appropriatecombination of fluorescent molecules only one excitation wavelength maybe required to detect the presence of both fluorescent moieties in closeproximity through the process of fluorescent energy transfer, whereinthe excitation wavelength excites one moiety which emits at theabsorption wavelength of the second moiety. Such simultaneous use willallow the detection of interaction of various transcription factors andcofactors with each other and with the DNA to activate and/or represstranscription from a specific regulatory sequence.

Cells for use in the present methods are cells having a plurality ofcopies of the nucleic acid in an array such that the nucleic acid can bedirectly detected when bound by fluorescently labeled compound. Suchcells are described herein and can be prepared as described herein. Anexample of such a cell is cell line 3134, having about two hundredcopies of the MMTV LTR-Ha-v-ras-gene, each of which has four copies ofthe binding site for glucocorticoid receptor, in direct tandem repeatsintegrated into the genome of the cell. Cells further can have a nucleicacid encoding a chimeric protein comprising a fluorescent protein fusedto the binding compound/transcription factor-of interest, i.e., thebinding compound/transcription factor which may bind the nucleic acid inthe array in the cell. For example, 3134 cells containingpCI-nGFP-C656G, pCI-nGFP-C656G, pTET-nGFP-C656G or pOP-nGFP-C656G areexemplified herein. Nucleic acids encoding a chimeric protein can eitherbe integrated or not, as best suits the specific method being performed.

The present invention also provides a method of characterizing cells inwhich a compound fluorescently labeled by a fluorescent protein isexpressed in the cell and the localization of the fluorescent proteinobserved for perturbation in localization of the fluorescently labeledprotein in the absence or presence of signals that affect proteinfunction (example of GFP-ER in the MCF7 and MDA-MB-231 cell line).

The present invention also provides a method of screening for a ligandthat activates gene targeting of a steroid receptor in the nucleus of amammalian cell comprising contacting the ligand with a cell having aplurality of steroid receptor response elements in an array such thatthe response element can be directly detected when bound byfluorescently labeled steroid receptor and the cell further comprising anucleic acid encoding a chimeric protein wherein a fluorescent proteinis fused to the steroid receptor; and directly detecting the location offluorescence within the cell, fluorescence aggregated at the site of thesteroid receptor response element array in the nucleus indicating aligand that activates the gene targeting of a steroid receptor in thenucleus of a mammalian cell. A ligand for any steroid receptor can bedetermined by this method by creating an array of the steroid receptorresponse element in the cell used. For example, cell line 3134 can beused to detect ligands that activate gene targeting of glucocorticoidresponse element, progesterone receptor, or aldosterone receptor. Anexample of a chimeric protein for use in this method in, for examplecell line 3134, is one that has a green fluorescent protein, such asAequorea fluorescent protein, fused to the N-terminus of glucocorticoidreceptor, such as the chimeric protein comprising the amino acidsequence set forth in SEQ ID NO: 2.

The present invention further provides a method of screening for aligand that activates the translocation of a steroid receptor to thenucleus or redistribution of a steroid receptor in a mammalian cellcomprising contacting the ligand with a cell having a plurality ofsteroid receptor response elements in an array such that the responseelement can be directly detected when bound by fluorescently labeledsteroid receptor and the cell further comprising a nucleic acid encodinga chimeric protein wherein a fluorescent protein is fused to the steroidreceptor; and directly detecting the location of fluorescence within thecell, change in the location of the fluorescence (e.g., cytoplasm tonucleus in the case of the glucocorticoid receptor or redistributionwithin each cellular compartment; e.g., for primarily nuclear receptors,such as the progesterone receptor, aggregation on the MMTV LTR-array inthe 3134 cell) could indicate a potential ligand of the receptor. In thepresent method, redistribution of the receptor can be directly observed.Also, in the case of the glucocorticoid receptor, translocation to thenucleus, even in the absence of binding to the nuclear DNA, can be seen,and importantly, one can see if a ligand causes only translocation tothe nucleus (by location of fluorescence primarily in the nucleus, butin a diffuse or reticular, rather than aggregated, pattern) or causesboth translocation to the nucleus and binding to nuclear DNA (bylocation in the nucleus aggregated primarily at a site). In the case ofestrogen receptor which has been partially activated due to traceestrogenic substances present in the culturing media, the receptor isnuclearly localized but shows dramatically different intranucleardistribution in two human breast cancer cell lines. In the case of MCF7cell, a human adenocarcinona breast cell line which contains endogenousestrogen receptor and shows hormone dependent growth, the GFP-taggedestrogen receptor is collected upon nuclear structures and shows focalaccumulation patterns. In contrast, the MDA-MB-231 cell, a humanadenocarcinoma breast cell line which lacks endogenous estrogen receptorand shows hormone-independent growth, the GFP-tagged estrogen receptor

under the same culturing condition is extremely diffuse. These twodramatic differences in the nuclear localization patterns in twodifferent human breast cancer cell lines suggests potential usefulnessof GFP-ER as a diagnostic reagent for characterizing

different human breast cancer cells as well as characterizing theprogression of human breast cancer. These differences in GFP-ERlocalization patterns also suggest an additional requirement forcelllular components in permitting the targeting of the estrogenreceptor onto nuclear structures; these cellular components maybe absentas the cell progresses from hormone-dependent to a hormone-independentstage in the progression of human breast cancer.

The present invention additionally provides a method of detecting in abiological sample the presence of an agonist of a steroid receptorcomprising contacting the sample with a cell having a plurality ofsteroid receptor response elements in an array such that the responseelement can be directly detected when bound by fluorescently labeledsteroid receptor and the cell further comprising a nucleic acid encodinga chimeric protein wherein a fluorescent protein is fused to the steroidreceptor; and directly detecting the location of fluorescence within thecell, the location of fluorescence aggregated at the site of the steroidreceptor response element array in the nucleus indicating the presenceof an agonist of the steroid receptor in the sample.

Biological samples can include any relevant sample from the body, suchas blood, plasma, urine and saliva.

The present invention also provides a method of detecting in abiological sample the presence of an antagonist of a steroid receptorcomprising contacting the sample and both agonist and antagonist of thesteroid receptor with a cell having a plurality of steroid receptorresponse elements in an array such that the response element can bedirectly detected when bound by fluorescently labeled steroid receptorand the cell further comprising a nucleic acid encoding a chimericprotein wherein a fluorescent protein is fused to the steroid receptor;and directly detecting the location of fluorescence within the cell, theabsence of fluorescence substantially aggregated at the site of thesteroid receptor response element array in the nucleus indicating thepresence of an antagonist of the steroid receptor in the sample.

Also provided is a method of monitoring the level of an agonist of asteroid receptor in a subject comprising periodically obtaining abiological sample from the subject, contacting the sample with a cellhaving a plurality of steroid receptor response elements in an arraysuch that the response element can be directly detected when bound byfluorescently labeled steroid receptor and the cell further comprising anucleic acid encoding a chimeric protein wherein a fluorescent proteinis fused to the steroid receptor; and directly detecting the location offluorescence within the cell, a decrease in fluorescence aggregated atthe site of the steroid receptor response element in the nucleus in alater-obtained sample relative to an earlier-obtained sample indicatinga decrease in level of the steroid agonist of the steroid receptor inthe sample and an increase in fluorescence aggregated at the site of thesteroid receptor response element in the nucleus in a later-obtainedsample relative to an earlier-obtained sample indicating an increase inlevel of the steroid agonist of the steroid receptor in the sample.

Further, provided by the present invention is a method of monitoring thebalance between levels of an agonist of a steroid receptor and anantagonist of the steroid receptor in a subject comprising periodicallyobtaining a biological sample from the subject, contacting the samplewith a cell having a plurality of steroid receptor response elements inan array such that the response element can be directly detected whenbound by fluorescently labeled steroid receptor and the cell furthercomprising a nucleic acid encoding a chimeric protein wherein afluorescent protein is fused to the steroid receptor; and directlydetecting the location of fluorescence within the cell, an increase influorescence aggregated at the site of the steroid receptor responseelement in the nucleus in a later-obtained sample relative to anearlier-obtained sample indicating an increase in level of the steroidagonist relative to level of the steroid antagonist in the sample, and adecrease in fluorescence aggregated at the site of the steroid receptorresponse element in the nucleus in a later-obtained sample relative toan earlier-obtained sample indicating an increase in level of thesteroid antagonist of the steroid receptor relative to level of thesteroid agonist in the sample.

The present invention also provides a method of determining an effectivedosage of a steroid receptor agonist in a subject comprisingtransferring into a set of cells from the patient a nucleic acidencoding a chimeric protein comprising a fluorescent protein fused to asteroid receptor; contacting the cells in the set with one of a selectedrange of dosages of the steroid agonist; and directly detecting locationof fluorescence in the set of cells, a dosage capable of locatingfluorescence substantially in the nucleus indicating an effective dosageof steroid receptor agonist.

Further provided by the present invention is a method of determining aneffective dosage of a steroid receptor agonist to maintain steroidreceptor activation for a selected period of time in a subjectcomprising administering to the subject a dosage of the steroid receptoragonist; periodically obtaining a biological sample from the subject;contacting the sample with a mammalian cell having a plurality ofsteroid receptor response elements in an array such that the responseelement can be directly detected when bound by fluorescently labeledsteroid receptor and the cell further comprising a nucleic acid encodinga chimeric protein wherein a fluorescent protein is fused to the steroidreceptor; and directly detecting the location of fluorescence within thecell, a dosage that maintains the location of fluorescence at the siteof the steroid receptor response element array in the nucleus for theselected period of time indicating an effective dosage.

The present invention additionally provides a method of detecting adefect in a response pathway of a steroid receptor in a subjectcomprising transferring into a cell from the subject a nucleic acidfunctionally encoding a chimeric protein comprising a fluorescentprotein fused to the steroid receptor and detecting the location offluorescence within the cell as compared to the location of fluorescencewithin a normal, control cell transfected with the nucleic acid, adifference in location of fluorescence within the cell of the subject ascompared to location of fluorescence within the normal, control cellindicating a defect in the response pathway of the steroid receptor.

The present invention also provides a method of determining whether adefect in a response pathway of a steroid receptor in a subject is intranslocation of the steroid receptor to a cell nucleus, comprisingtransferring into a cell from the subject having the defect a nucleicacid functionally encoding a chimeric protein comprising a fluorescentprotein fused to the steroid receptor and detecting the location offluorescence within the cell, the location of fluorescence substantiallyin the cytoplasm of the cell indicating the defect is in translocationof the steroid receptor to the nucleus.

The present invention also provides a method of characterizing aligand's effect on cellular localization of a compound to which theligand binds in a cell comprising contacting the ligand with a cellhaving the compound fluorescently labeled by a fluorescent protein anddirectly detecting the location of fluorescence within the cell, thelocation of fluorescence in the cell indicating the localization effectof the ligand on the compound. Compounds can be, e.g., steroidreceptors, transcription factors and the like. For example, the examplesprovide characterization of localization of GR in response to twoligands, dexamethasone and RU486, and characterization of thelocalization of ER in response to ligands, agonist beta-estradiol oranti-estrogens, 4-hydroxytamoxifen or ICI164384.” By this method, theligands triggering binding of so-called “orphan receptors” to theirbinding site(s) can be discovered.

Additionally provided is a method of determining a binding site for aDNA-binding protein comprising contacting the DNA-binding proteinfluorescently labeled by a fluorescent protein with a cell having aplurality of copies of a nucleic acid having a putative binding site inan array such that the putative binding site can be directly visualizedwhen bound by the fluorescently labeled DNA-binding protein, anddirectly detecting the location of fluorescence within the cell, thepresence of fluorescence aggregated at the putative binding siteindicating a binding site to which the DNA-binding protein binds. Theabsence of fluorescence aggregated at the putative binding site cansuggest a binding site to which the DNA-binding protein does notsignificantly bind.

the location of fluorescence in the cell indicating the localizationeffect of the ligand on the compound.

Also provided by the present invention is a method for screening forgene-specific combinations of compounds that bind the gene specifically,comprising contacting (a) a first compound labeled by a fluorescentprotein emitting a first spectrum of light and a second compound labeledby a fluorescent protein emitting a second spectrum of light with (b) acell having a plurality of copies of the regulatory region of the genein an array such that the regulatory region can be directly detectedwhen bound by compound labeled by a fluorescent protein. Fluorescencefor the first and second spectrum is then localized. Aggregation offluorescence of both the first and second spectrum at the site of theregulatory region array would indicate a combination of compounds thatbinds the gene specifically; the location of only one spectrumaggregated at the array would indicate that only the correspondingcompound binds the gene of interest directly. By using a combination ofscreens, compounds that bind the specific DNA both directly andindirectly can be determined for a gene of interest. Such gene-specificcombinations of compounds can be used to develop gene-specific drugsthat interfere with transcription activators in a selective manner. Thismethod is based on the fact that each transcription factor, cofactor,etc. affects many genes, but for each gene there is likely only onecombination of factors and cofactors that activates/represses it.Therefore, once, by this screening method, it has been determined whichcombination of factors causes activation or repression of a specificgene, then a combination of drugs, to affect all relevant factors forthat specific gene, can be administered to selectively activate/repressthat gene. Thus a combination of drugs can ultimately be used toactivate or repress the selected gene.

Statement Concerning Utility

The present invention provides methods for directly detecting thebinding of compounds to nucleic acids. The present invention allows asimple and straightforward manner in which direct interaction between asequence-specific DNA binding protein or its co-factor and its putativeregulatory site in the in vivo genomic context can be addressed. Forexample, GFP-steroid fusion proteins and cell lines containing receptorbinding sites (response elements) in multimerized arrays are providedfor direct visualization of in vivo gene targeting. These reagentsprovide a simple, rapid, straightforward, sensitive, and biologicallyrelevant assay for each target nucleic acid and binding compound. Thesereagents can in turn be used for several medically importantapplications, including diagnostic tests for concentration of cognateligand in clinical samples (urine, saliva, blood, etc.). Additionally,the reagents can be used for direct tests for defects in steroidpathways in subjects. The reagents further can be used for screening ofchemical banks for compounds with ligand agonist activity for eachreceptor, and development of drugs based on these activities.Furthermore, using the fluorescent protein-steroid receptor fusions,colocalization of a selected receptor with any other cofactor that maybe recruited to the chromosome template can be evaluated. Using aseparate tag (different color) for the DNA target, the steroid receptorcan be fused to any factor that may be recruited by the steroid receptorand determine if the factor colocalizes on the DNA target when thesteroid receptor is activated, allowing for a direct test for defects infactor colocalization in human disease/syndromes. This ability toobserve direct interaction of any trans-regulatory factor or co-factorand its regulatory site in vivo provides a screening method for usefuland novel drugs, directed against trans-regulators, and the developmentof gene-specific multi-drug therapies. Using the glucocorticoid receptoras an example and known ligands of the receptor (e.g., agonistdexamethasone and antagonist RU486), the validity of the methodology isherein demonstrated. In a specific example, the ability to monitoreffective hormone concentration by this novel methodology in real timecan lead to the development of diagnostic kits that can be used toproperly gauge the required amounts of hormone administered to patientsrequiring long-term or short-term hormone treatment. In addition, in thecase of those trans-acting factors regulated at the level of thenuclear/cytoplasmic and cytoplasmic/nuclear translocation step, diseasesarising from such a failure can be directly diagnosed by fusion of theregulatory molecule to a fluorescent moiety using standard recombinantDNA techniques. With this simple, inventive methodology, novel classesof drugs directed not only against members of thesteroid-ligand-dependent transcription factors but to new classes ofdrugs that target other transcription factors or their co-factors can bescreened. Furthermore, by using combination of drugs which targetcertain trans-regulatory factors either specifically or selectively, agene-specific based drug therapy regimen can be created. This multi-drugtherapy designed against a certain critical gene implicated for aparticular human disease would be tailored to affect the activity of thetrans-regulatory factors all of which act synergistically to regulatethe transcription of the gene implicated in the disease. Furthermore,the reagents allow the development of transgenic animals containingfusion proteins such as each of the GFP-receptor fusions that canprovide a unique tool to study subcellular distribution of the receptorsin all tissues of the animal, and the effect of pharmacologic agents onfunction of each of the receptors in the various tissues. Numerous otherutilities will be apparent to the skilled artisan in light of thepresent invention.

EXAMPLES

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

Plasmids.

pCI-nGFP-C656G: Plasmid pCI-nGFP-C656G was derived from pCI-nH6HA-C656G(Smith et al., submitted) and pZA69 (a kind gift from Mike Moser andRavi Dahr). pZA69 contains a BspHI fragment of pZA66, a plasmidcontaining S65T GFP (Tsien, R. Y. (1995) Nature 373,663-664) with theinternal NcoI site removed by a silent mutation. The pCI-nH6HA-C656G DNAexpresses the rat glucocorticoid receptor with the C656G mutation(kindly provided by S. Simons, Jr.) under the control of the CMVpromoter/enhancer, and is tagged at the N-terminus with (his)₆ andhemagglutinin epitope recognized by monoclonal antibody 12CA5 (Niman, H.L., Houghten, R. A., Walker, L. E., Reisfeld, R. A., Wilson, I. A.,Hogle, J. M. & Lemer, R. A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80,4949-4953); this DNA was cleaved at a unique site with PvuII, separatingthe two tags from the rest of the glucocorticoid receptor. A 768 bp DNAfragment containing the GFP cDNA with thq S65T mutation was inserted atthe PvuII site. This GFP DNA fragment is obtained by digesting pZA69with BglI, attaching a BglI hairpin linker with the sequence(5′-GCGCGCTGATCAGAATTCCTTTTAGGAATTCTGATCAGCGCGCTGA-3′) (SEQ ID NO:3),recutting the resulting DNA with BclI and XhoI, and then filling-in withthe large fragment of DNA polymerase (Klenow) to create a 768 bpblunt-end fragment.

pCI-nGL1-C656G: The GFP (S65T) variant with the jellyfish codon usage inthe mammalian expression GFP-GR vector, pCI-nGFP-C656G, has beenreplaced with a humanized codon usage from the plasmid, pGreenLantem-1(LifeTechnologies, Inc., Gaithersburg, Md., catalog number 10642-015),for improved translation in mammalian cells [this humanized GFP is alsoapproved for in vitro diagnostic use by LifeTechnologies] to generate animproved mammalian expression GFP-GR plasmid, pCI-nGL 1-C656G.

pCI-nGL2-C656G; pCI-nGL3-C656G; pCI-nGL4-C656G; pCI-nGL5-C656G;pCI-nGL7-C656G; pCI-nGL9-C656G; pCI-nGL10-C656G; pCI-nGL11-C656G: Bluevariants of GFP (BFP) in a humanized codon usage (improved bluevariants) have been generated which are fused to the rat glucocorticoidreceptor (C656G). Each has a different chromophore. Site-directedmutagenesis of the GFP element of pCI-nGL1-C656G (which has humanizedGFP) was performed to introduce known chromophore-altering mutationsinto the GFP element. Site-directed mutagenesis was performed usingChameleon™ double-stranded site-directed mutagenesis kit (Stratagene,catalog number 200509). These new fusion plasmids were then expressed in1471.1 cells. Fluorescent spectrophotometry indicates the chromophoresare present and that the GFP-GR fusion is intact. Additional mutants,such as those producing a longer wavelength chromophore, e.g. redchromophores, can be made by the same methods.

GFP-ER plasmid: The glucocorticoid receptor portion of pCI-nGL1-C656GDNA has been replaced with a human estrogen receptor (ER). ER binds toan ER response element in cells. The GFP-ER was found to be functionalin both transcriptional activation as well as proper subcellularlocalization in several cell lines, as described below. GFP-ER plasmidis made using the same site-directed mutagenesis as for preparing bluevariant plasmids. Briefly, a cDNA encoding ER is inserted intopCI-nGL1-C656G in place of the GR cDNA. An ER cDNA (Green, Stephen, etal., Nature 320:134-139 (1986) (GenBank accession number X03635) (note:this sequence has a Gly₄₀₀ to Val₄₀₀, mutation); Greene, Geoffrey L., etal. Science 231:1150-1154 (1986) (GenBank accession number M12674(having Gly₄₀₀ to Val₄₀₀ mutation)); Pfeffer, U. Cancer Res. 53:741-743(1993) (GenBank accession number X73067) (ER fragment having correctGly₄₀₀, region coding sequence)) is mutagenized to create a MluI site atthe start point of translation of ER and a SalI site in the 3′untranslated region of the cDNA (alternatively, the ER sequence can begenerated by PCR). pCI-nGL1-C656G has a unique BssHII site after the(gly-ala)5 linker and a SalI site after the GR portion of the plasmidDNA. The DNA cut end made by MluI is complementary to the end made bythe BssHII. Therefore, the GR cassette can be removed by BssHII/SalIdigestion and the ER cassette (released by MluI/SalI digestion)subcloned into the remaining vector at the BssHI and SalI cut ends.

pOP-nGFP-C656G: The original cDNA encoding GFP-GR from the plasmid,pCI-nGFP-C656G, has been subcloned into a tetracycline-regulatablemammalian expression vector, pTET-Splice (LifeTechnologies, catalognumber 10583-011), to give pTET-nGFP-C656G, and a lac-regulatablemammalian expression vector, pOPRSVI CAT (Stratagene Cloning Systems,catalog number 217450) to give pOP-nGFP-C656G.

Other plasmids used in this study are: pLTRLuc (full-length MMTV LTRdriving the expression of a luciferase gene) (Lefebvre, P., Berard, D.S., Cordingley, M. G. & Hager, G. L. (1991) Mol. Cell. Biol. 11(5),2529-2537), pCMVIL2R (IL2R expression plasmid) (Giordano, T., Howard, T.H., Coleman, J., Sakamoto, K. & Howard, B. H. (1991) Exp Cell Res. 192,193-197), and pUC18 (Life Technologies, Inc.).

GFP-fusion plasmids of the invention are tested for expression andsubcellular localization by transfection into several mammalian cells.For example, GFP-GR plasmids were analyzed in C 127 (mouse) cells, HeLa(human cervical cancer) cells, and MCF7 (human breast adenocarcinoma)cells (ATCC accession number HTB22). GFP-ER plasmids were analyzed in1471.1 cells, C127 (mouse) cells, MCF7 (human breast adenocarcinoma)cells, and MDA-MB-231 (human breast adenocarcinoma) cells (ATCCaccession number HTB 26). Localization is observed in the absence ofadded hormone in either 5% or 10% charcoal-stripped fetal calf serum.Cells having GFP-fusion plasmids are then treated with a selectedligand, and subcellular localization and quantitative observations aremade.

Cell Line 1471.1 and Derivatives

Cell line 1471.1 contains multiple copies of a BPVMMTV-LTR-chloramphenicol acetyltransferase (CAT) reporter gene fusionintroduced in the murine adenocarcinoma C127 cell (Archer, T. K.,Cordingley, M. G., Marsaud, V., Richard-Foy, H. & Hager, G. L. (1989) inProceedings: Second International CBT Symposium on the Steroid/ThyroidReceptor Family and Gene Regulation, eds. Gustafsson, J. A., Eriksson,H. & Carlstedt-Duke, J. (Birkhauser Verlag AG, Berlin), pp. 221-238).

Derivatives of 1471.1 cells which contain over a thousand copies of theMMTV LTR-CAT have also been generated with the tetracycline- andlac-regulatable GFP-GR (pTET-nGFP-C656G and pOP-nGFP-C656G), e.g., cellline 3677. In these derivative cell lines, GFP-GR expression occurs upontetracycline withdrawal from 5 ug/ml or upon induction with IPTG usingstandard procedures and as recommended by the manufacturer of theseinducible systems.

Transfection. Plasmid DNA was transiently introduced into 1471.1 cellseither by calcium phosphate coprecipitation using a BES-based buffer(Chen, C. & Okayama, H. (1987) Mol. Cell Biol. 7, 2745-2752) or byelectroporation. For calcium phosphate coprecipitation, semi-confluentcells maintained in Dulbecco's modified eagle media (DMEM; LifeTechnologies, Inc.) supplemented with 10% fetal calf serum (FCS; LifeTechnologies, Inc.), 2 mM glutamine, and 50 mg/ml gentamicin sulfatewere trypsinized, washed, resuspended at 7×10⁴ cells/ml in DMEMsupplemented as above except FCS was treated withcharcoal/dextran-treated fetal bovine serum (Hyclone Laboratories,Inc.), dispensed as 1 ml into 2×2 cm² Lab-Tek Chamber Slide (Nunc, Inc.)or as 10 ml into a 100 mm petri dish layered with 24.5 mm-diameterDvorak-Stotler coverslips (Nicholson Precision Instrument). Cells weregrown overnight in a 37° C. humidified incubator with 5% CO₂. Thefollowing morning, media was replaced with fresh supplemented DMEMcontaining dextran/charcoal treated FCS, and in the afternoon, cellswere transfected with 1 ml of transfection mixture containing 20 μgplasmid DNA (as indicated in the appropriate figure legend) per 10 ml ofcells, essentially as described (Chen, C. & Okayama, H. (1987) Mol. CellBiol. 7, 2745-2752). Cells were left overnight in a 37° C. humidifiedincubator with 2.9% CO₂. About 12-16 hours after transfection, media wasreplaced, and the cells were allowed to recover for two hours beforefurther treatment and imaging. For calcium depletion experiments, cellswere electroporated with 5-20 μg pCI-nGFP-C656G DNA for 2×10⁷ cells in0.2 ml cold DMEM at 250 V and 800 μF., left to recover on ice for 5minutes, and then diluted in DNEM supplemented with dextran/charcoaltreated FCS before plating. Cells were then grown for 12 to 16 hours ina 37° C. humidified incubator at 5% CO₂. Before treatment and imaging,cells were fed with fresh media.

Enrichment of Transfected Cells and Analysis of Cytosolic Extracts.Cells that took up exogenous DNA were enriched by cotransfection withpCMVIL2R, an IL2R (interleukin 2 receptor) expression plasmid, andselection for IL2R+cells using magnetic beads (Dynal) coated with mouseanti-human IL2R antibody (Boehringer Manneheim, clone 3G10), asdescribed (Giordano, T., Howard, T. H., Coleman, J., Sakamoto, K. &Howard, B. H. (1991) Exp Cell Res. 192, 193-197). Extracts from theIL2R⁺ and IL2R⁻ cells were made by three cycles of freezing and thawingof the cell suspension in either 100 mM sodium phosphate (pH 7.8) with 1mM DTT or 250 mM Tris-HCl (pH 7.8). After clarifying the lysate,extracts made with the phosphate buffer was used to assay for the amountof luciferase activity in a MicroLumat LB96P as recommended by themanufacture, EG&G Berthold. For the Tris-buffered extract, CAT activitywas assayed as described (Gorman, C. M., Moffat, L. F. & Howard, B. H.(1982) Mol. Cell Biol. 2, 1044-1051). Protein concentration wasdetermined by the method of Bradford using the Bio-rad Protein Assayreagent (Bio-rad Laboratories, Inc.).

Determination of Intracellular Calcium. Intracellular free calciumconcentrations were determined in single cells by measuring the signalfrom the calcium sensitive indicator Fura-2, according to Tsien andHarootunian (Tsien, R. Y. & Harootunian, A. T. (1990) Cell Calcium 11,93-109). Briefly, cells were cultured on cover slips and electroporatedwith the GFP-GR chimera one day before microscopy. In preparation forimaging, cells were treated for 30 min with either assay buffer (Hank'sbalanced salt solution without phenol red, with 2 mg/mil glucose and 1mg/ml BSA, containing 3 mM Ca⁺⁺) or with calcium-free buffer (Eagle'sNo. 2 medium without calcium, containing 1 mg/ml BSA, 5 mM EGTA, 5 μMthapsigargin, 2 μM ionomycin). The cells were then loaded with 5 μmol/LFura-2-AM (from Molecular Probes Inc.) and 0.02% pluronic F-127 witheither calcium-free or calcium supplemented media (30 min at roomtemperature, washed three times, then incubated for 15 min at 37° C.).After loading, cells were placed into a Dvorak-Stotler chamber (innervolume 224 μl) and perfused at 37° C. with either calcium-containing orcalcium-free media. Intracellular calcium content was measured in threeindependent experiments—at least 20 cells in each experiment. Ratioimaging was performed using Image 1 software (Universal Imaging Corp.)running on an IBM PC, using 340 nm and 380 nm excitation, 510 nmemission, and 490 nm dichroic barrier filters, a Zeiss PhotomicroscopeIII microscope, enclosed into a temperature controlled incubator, and anintensified (Videoscope) CCD camera (Dage 72), and optical disc recorder(Panasonic). The system was calibrated for [Ca²⁺]_(i) measurement usingFura-2 pentapotassium salt and calibration buffer kit from MolecularProbes Inc. Intracellular free calcium concentrations in cells withcalcium supplemented buffer were 350±183 nM, while in calcium-freebuffer 60±11 nM.

Image Acquisition and Analysis. For time course studies, cells wereplaced into a Dvorak-Stotler chamber (inner volume 224 μl) and perfusedat 37° C. with assays buffer for 3 min, then with the same buffercontaining 1 nM dexamethasone (dex) for 2 hours at 10 ml/hour flowrates. Samples were evaluated using a Zeiss Axiovert 10 microscopesurrounded by an incubator and equipped for epifluorescence with.illumination from XBO burner, 480 nm excitation and 535 nm emission and505 nm dichromatic barrier filters (from Chroma Technology Corp.).Images were acquired every 15 seconds with a high resolution, cooled CCDcamera equipped with an electromechanical shutter (Photometrics p200).Images were collected on Silicon Graphics workstations (4D310-VGX),using custom software, incorporating functions from a vendor suppliedlibrary (G. W. Hannaway & Assoc.). Experiments requiring real time imageacquisition were performed on the imaging system described for theintracellular calcium measurements.

Confocal laser scanning microscopy was carried out on a Nikon Optiphotmicroscope equipped with BioRad MRC-600 confocal laser scanning unit,with fluorescent excitation produced by the 488 nm line of akrypton-argon laser, and using a Fluor 100/1.3 oil phase objective. Fromliving cells expressing GFP-GR, serial 0.5 μm optical sections werecollected and the digitized images were imported into a Silicon GraphicsIndigo 2 workstation. Three dimensional image rendering, analysis andreconstruction was carried out with the ANALYZE software from the MayoClinic.

Tagging of a Highly Dexamethasone-Sensitive Form of GR with a HighlyFluorescent Variant of GFP. To develop a highly efficient, fluorescentversion of the glucocorticoid receptor, we generated a GFP-GR chimera inwhich cDNA encoding a 27 kDa GFP variant is fused in frame to the secondamino acid of a rat glucocorticoid receptor (FIG. 3A). The GFP variantcontains a serine to threonine substitution at amino acid 65 (S65Tmutation) from the jellyfish Aequorea Victoria, which increases theefficiency of formation of the GFP chromophore by accelerating the rateof oxidation required for chromophore generation. In addition, theresulting chromophore is six-fold more fluorescent than the wild-typeGFP (Heim, R., Cubitt, A. B. & Tsien, R. Y. (1995) Nature 373, 663-664),making the use of this chromophore perhaps the most sensitive method forlabelling proteins (Wang, S. & Hazelrigg, T. (1994) Nature 369,400-403). Additionally, the chromophore is formed faster, potentiallyexplaining why expression at 37° C. in mammalian cells is achievedherein, contrary to reports that the GFP chromophore does not form at arelatively high temperature of 37° C. (Ogawa, et al. Proc. Natl. Acad.Sci. USA 92:11899-11903 (1995)).

Since glucocorticoid receptor is ubiquitously present in all mouse cellsand selective activation of the tagged receptor is required to assessthe functionality of the receptor, we therefore used a glucocorticoidreceptor having a higher affinity for its ligand than the endogenousreceptor. To this end, S65T GFP was fused to a rat glucocorticoidreceptor that contains a cysteine to glycine mutation at position 656 ofthe steroid binding domain (Chakraborti, P. K., Garabedian, M. J.,Yamamoto, K. R. & Simons, S. S. J. (1991) J. Biol. Chem. 266,22075-22078). This point mutation, C656G, increases the affinity of thereceptor ten-fold for its ligand. A dose response curve shows completeactivation of GFP-GR at 1 nM dexamethasone and half maximum at 0.1 nM;the endogenous mouse receptor is fully activated at 100 nM dexamethasonewith the half maximal stimulation at 10 nM. Thus, presence of the C656Gmutation permits selective activation of the transfected chimericreceptor without activation of the endogenous receptor.

Transcriptional Competence of GFP-GR. When the plasmid encoding thischimera, pCI-nGFP-C656G, is introduced into cultured mouse cells, afusion polypeptide with the predicted molecular weight of 118 kDa isproduced. When these cells are stimulated with 1 nM dexamethasone, aco-transfected reporter construct (pLTRLuc) containing the luciferasereporter gene under the control of the mouse mammary tumor viruspromoter (MMTV LTR) is activated (FIG. 3B). In addition 1 nMdexamethasone-treated cells show accumulation of luciferase activitydependent on the amount of GFP-GR expression plasmid included in thetransfection. In the absence of any GFP-GR expression plasmid, nosignificant luciferase activity accumulated in the 1 nMdexamethasone-treated cells, indicating that 1 nM dexamethasoneactivated the GFP-GR chimeric protein but not the endogenous GR. Withincreasing amount of the GFP-GR expression plasmid, luciferase activityin the 1 nM dexamethasone-treated IL2R⁺ cells reaches the same level asthat in 100 nM dexarnethasone-treated IL2R⁺ cells lacking GFP-GRexpression plasmid (compare 2 μg pCI-nGFP-C656G, 1 nM dex with 0 μgpCI-nGFP-C656G, 100 nM dex). Since the 100 nM dexamethasone treatmentgives complete activation of the endogenous GR in the latter case, weconclude that the GFP-GR chimeric receptor is fully functional indexamethasone-mediated transcriptional activation of the transientlyintroduced reporter plasmid DNA.

Furthermore, derivative cell lines of 1471.1 which contain over athousand copies of the MMTV LTR-CAT have also been generated with thetetracycline- and lac-regulatable GFP-GR (pTET-nGFP-C656G andpOP-nGFP-C656G), allowing GFP-GR expression upon tetracycline withdrawalfrom 5 ug/mil or upon induction with IPTG using standard procedures andas recommended by the manufacturer of these inducible systems. In thesecells, acceptable levels of GFP-GR is reached after overnight inductionof the regulatable promoters.

Assay for Ligand Effects: GFP-fusion plasmids of the invention areassayed for effects of a selected ligand on subcellular localization bytransfection into selected cells. C127 (mouse) cells, HeLa (humancervical cancer) cells, and MCF7 (human breast adenocarcinoma) cells(ATCC accession number HTB22) were each transfected with GFP-GR plasmidsand with GFP-ER plasmids. Localization is first observed in the absenceof added hormone in either 5% or 10% charcoal-stripped fetal calf serum.Cells having GFP-fusion plasmids are then treated with a selectedligand, and subcellular localization and quantitative observations aremade.

GFP-GR Results: GR is observed to be localized to the cytoplasm in theabsence of added ligand. However, when cells having the GFP-GR plasmidswere treated with dexamethasone, foci of fluorescent signal (massivevariation in the peak and valley of fluorescent signal) were observed inthe nucleus (see, e.g., FIG. 4(A)). When these cells were treated withRU486, the fluorescent signal is near uniform, with a matrix-likeappearance where fluorescent signal appears (see, e.g., FIG. 4(B)).

GFP-ER Results: ER is observed to be localized to the nucleus in theabsence of added estrogen hormone. When a hormone-dependent, estrogenreceptor positive human breast cancer cell line, MCF7, is transfectedwith GFP-ER expression plasmid, expression

of GFP-ER results in nuclear signal that is structured with peaks andvalley of concentration of signals (FIG. 5(A)). Although the GFP-ER isalready partially activated due to trace estrogenic substances presentin the culturing medium, addition of agonist beta-estradiol leads tofurther concentration of nuclear signals on nuclear structures. In thecase of a hormone-independent, estrogen receptor negative human breastcancer cell line, MDA-MB-231, the expressed GFP-ER in the absence ofadded ligand shows a diffuse, fuzzy pattern, with only hints ofattachments to structures in the nucleus.

These results provide an example of use of the present method as adiagnostic for missing, dysfunctional or nonfunctional components in anyselected cell. For example, upon determining that a ligand, such asestrogen for ER binding to its response element, is present in a cancercell and used for growth or maintenance of the cell, one can treat thecancer by administering a compound to deprive the cell of that ligand,such as by administering anti-estrogen to a breast cancer showing thesame results as MCF7 (hormone-dependent) to reduce growth of the cells.Similarly, such a diagnostic can tell one if such a treatment in anothercell would be futile because the cell is not dependent upon that ligand.

These results also demonstrate that the present method can be used as ascreen to classify cell types for the ability (or lack of ability) totarget nuclear structures, to traffic compounds in a particular pattern,etc. This also provides information for selecting treatment regimens forvarious diseases or disorders, based on activating, inactivating oraltering the function of ligands in the cell.

Transcriptional activation of the MMTV LTR target genes by GFP-GR showsa ligand specificity characteristic of glucocorticoid receptor. Whenactivated by dexamethasone, GFP-GR is competent to induce not only thetransiently introduced MMTV LTR-luciferase reporter DNA, as mentionedabove (FIG. 3B), but also the multi-copy MMTV LTR-CAT reporter genespresent in 1471.1 cells (1 nM and 10 nM dex, FIG. 3C). In contrast,treatment with 10 nM RU486, an antagonist with little GR agonistactivity, or progesterone, a poor agonist, results in little activationof the MMTV LTR-CAT reporter; 17-β-estradiol, a steroid that shows noaffinity for GR, fails to activate the LTR. Thus, the ligand specificityof GR in the transcriptional activation of the MMTV LTR is maintained inGFP-GR expressing cells.

Visualization of GFP-GR Cytoplasm-to-Nuclear Translocation in a SingleMetabolically Active Cell. Because the S65T variant of the GFPchromophore is resistant to photobleaching (Heim, R., Cubitt, A. B. &Tsien, R. Y. (1995) Nature 373, 663-664), it was possible to useconfocal and time-lapse video microscopy to observe GFP-GR over extendedperiods. Using computer controlled high resolution video and confocallaser scanning microscopy, we examined transfected samples forsubcellular localization of the chimeric GFP-GR protein. We observedsignificant fluorescence in the cytoplasm of about ten percent of totalcells, approximately the fraction that typically acquires transfectedDNA. Thus, the GFP was functional as a chromophore in a majority of theexpressing cells in this mammalian system.

Upon exposure to dexamethasone, translocation of GFP-GR occurs in 100%of fluorescing cells, with the rate of cytoplasm-to-nucleartranslocation dependent on the concentration of hormone. At 10 nM,complete translocation was induced within 10 min at 37° C., with halfmaximal nuclear accumulation at 5 min; this rate is consistent withprevious findings (Picard, D. & Yamamoto, K. R. (1987) EMBO. J. 6,3333-3340). The rate of translocation is decreased with 1 nMdexamethasone (complete translocation over 30 min with half maximum at9-10 min) and further reduced with 0.1 nM dexamethasone (completetranslocation within 2 hours with half maximum at 1 hour).

Analysis of a time-lapse series revealed that GFP-GR accumulated alongfibrillar structures and in the perinuclear region very rapidly afterhormone addition, probably within seconds. Murine adenocarcinoma cellswere cultured on cover slips and transfected with GFP-GR fusion chimeraone day before microscopy. Cells were placed into a Dvorak-Stotlerchamber and perfused at 37° C. with assay buffer for 3 min, then withbuffer containing 1 nM dexamethasone for 2 hrs. With real-time imaging;perinuclear accumulation was observed in a pulsatile pattern with 1-2second intervals between brightness changes. GFP-GR accumulation wasmore intense along fibrillar structures in the perinuclear region. After3 min with 1 nM dexamethasone, GFP-GR was noticeably present in thenucleus, but not in the nucleoli. When approximately ⅓ of the proteinhad been translocated (9-10 min), a punctate pattern appeared, andtranslocation was complete after 30 min. During translocation, the cellsfrequently became rounded, and moved along the long axis of the cell. Weobserved reduction of the cell surface, as well as the nuclear volumeduring the translocation. One hour after hormone treatment, the cellsreattach and regain a more flattened shape.

Ligand Specificity of Cytoplasm-to-Nuclear Translocation. Cells weretreated with buffer (A), 10 nM 17-β-estradiol (B), 10 nM dexamethasone(C), or 10 nM RU486 (D) for 30 min at 37° C. At the end of hormonetreatment, images from living cells expressing GFP-GR were visualizedwith confocal laser scanning microscope as described above. Whilecomplete translocation of GFP-GR was observed in all fluorescing cellstreated with dexamethasone, other classes of steroid hormones inducedGFP-GR translocation to varying extent reflective of the affinity forGR. The glucocorticoid antagonist RU486, known to have a high affinityfor GR (Chakraborti, P K., Garabedian, M. J., Yamamoto, K. R. & Simons,S. S. J. (1991) J. Biol. Chem. 266, 22075-22078), was as potent asdexamethasone for induction of translocation. Progesterone, a weak GRagonist, required a concentration 100-fold higher than dexamethasone fortranslocation; however, approximately ½ of the GFP-GR remained in thecytoplasm. In contrast, 17-β-estradiol, a steroid hormone that does notbind GR, did not cause intranuclear GFP-GR accumulation (10 nM). Thus,GFP-GR maintained ligand-dependent cytoplasm-to-nuclear translocation,with analog specificity identical to that for the untagged GR with theC656G point mutation (Chakraborti, P. K., Garabedian, M. J., Yamamoto,K. R. & Simons, S. S. J. (1991) J. Biol. Chem. 266, 22075-22078).Furthermore, while ligand binding may suffice to trigger efficientcytoplasm-to-nuclear translocation, it may not necessarily cause bindingof the receptor to its nuclear target and it may cause varying degreesof activation of the target gene.Role of Intracellular Free Calcium and Energy in GFP-GR Translocation.Two important issues concerning the nuclear import of proteins were alsoaddressed; these include the role of Ca⁺⁺, and the energy requirement oftranslocation. Intracellular stores of Ca⁺⁺ were depleted by incubatingthe cells for 1 hour with the endoplasmic reticulum Ca⁺⁺-ATPaseinhibitor, thapsigargin, and the calcium ionophore, ionomycin, incalcium-free media (intracellular free calcium content was measured withratio imaging in Fura-2 loaded cells). The cytoplasmic pattern of GFP-GRwas not significantly altered by calcium depletion. When Ca⁺⁺-depletedcells were subsequently exposed to dexamethasone (10 nM for 30 min. at37° C.) in Ca⁺⁺-free media, the hormone induced complete translocationof GFP-GR, as seen by images taken from living cells with a confocallaser scanning microscope.

To study the energy dependence of ligand binding, cells were exposed todexamethasone (10 nM) at 4° C.; then hormone was removed and the cellswere warmed to 37° C. under continuous monitoring with video-microscopy(cooled CCD camera from Axiovert 10 microscope system). At 4° C.,translocation was completely arrested. Rewarming led to completetranslocation and reappearance of the focal GFP-GR localization. Thisexperiment indicates that hormone binding to GR in living cells does notrequire energy in contrast to the energy-dependent step oftranslocation.

Focal Accumulation of Nuclear GFP-GR Correlates with TranscriptionalActivation. When the intranuclear accumulation of GFP-GR is examined indetail, it is readily apparent that the receptor localizes mostprominently at specific foci within the nucleus. In addition, there is alow level of accumulation in a diffuse reticular pattern, forming thebasis for the nuclear background fluorescence. The number of these focalaccumulations are unique to dexamethasone-treated cells and are notobserved in 17-β-estradiol- or progesterone-exposed cells. InRU486-treated cells, focal points are not readily discernible. Instead,GFP-GR accumulates in a diffuse pattern with regions of condensation ina reticular pattern, such that regions of bright fluorescence appearthread-like in shape instead of as distinct foci. Depletingintracellular Ca⁺⁺ did not affect the dexamethasone-mediated formationof intranuclear foci. The ability of agonist to induce focalaccumulation of GFP-GR correlated strongly with its ability to activatetranscription (FIG. 3C). The striking accumulation ofdexamethasone-activated GFP-GR into intranuclear foci immediatelysuggests that a specific architecture may underlie this distribution. Tofurther examine the structure of intranuclear GR binding sites, confocallaser scanning fluorescent microscopy and three dimensional imagereconstruction was carried out.Organized Architecture of Interphase Nuclei as Revealed by GFP-GR.Three-dimensional architecture of GFP-GR nuclear target sites wasanalyzed. Serial 0.5 μm sections of nuclei from dexamethasone treatedcells were collected with confocal laser scanning fluorescentmicroscope, digitized images were imported into a Silicon GraphicsIndigo 2 workstation, and three dimensional image segmentation,rendering, analysis and reconstruction was carried out with the ANALYZEsoftware. GFP-GR distributions in the nuclei are displayed aspseudocolored, voxel-gradient-shaded, three dimensional projections.Three dimensional image analysis of the points of GR accumulation indexamethasone-treated cells reveals a non-random distribution of GFP-GRaccumulation. Most strikingly, comparison of adjacent cells demonstratesa reproducible pattern of intranuclear structure for GFP-GRaccumulation. A predominance of GFP-GR-accumulating foci is observed inthe quadrant of the nucleus adjacent to the glass attachment surface ofthe cell, while a group of large patches of GFP-GR-containing foci areobserved in the top half. Nucleolar structures were always devoid ofGFP-GR. The nuclear pattern of RU486-treated cells was again strikinglydifferent from dexamethasone-treated cells. Although essentially all ofthe GFP-GR is translocated, intranuclear RU486-liganded receptor isdistributed throughout the nucleus in a reticular pattern but excludingnucleoli.3134 Cell Line and Derivatives

The 3134 cell line was derived from a mouse line designated 904.1. Thiscell was established by transfection of a murine mammary carcinoma line(C 127) with a plasmid containing three functional segments: a) thebovine papilloma virus (BPV) 69% transforming fragment serves as areplicon in mammalian cells, b) the mouse mammary tumor virus (MMTV) LTRis a steroid responsive promoter and contains the GR binding sites, andc) the Ha-v-ras gene is a transforming oncogene and serves as a reporterfor the MMTV promoter.

This plasmid replicates in 904.1 cells as an 9 kb episomal circle.During passage of this cell line, a spontaneous integration eventoccurred. This event resulted in the integration of a tandem array ofthe BPV/MMTV-LTR/Ha-v-ras cassette in perfect head-to-tail orientation.The integrated structure is diagrammed in FIG. 2. Standard agarose gelelectrophoresis and southern transfer hybridization analysis with a BPVprobe (standard gel) was performed. CHEF gel high molecular weightanalysis, again with a BPV probe (CHEF gel) was also performed. When theintegrated array is digested with a one-cut restriction enzyme, therepeat unit of 8.8 kd is liberated as a single fragment (BamHI). NdeIand EcoRV (both no-cutters for the repeat unit) digestion leads to nodigestion in the standard gel; EcoRV liberates a fragment approximately2.2 megabase pairs in size. Enzymes that cut multiple times (PstI) giverise to the appropriate fragments for the repeat unit. Since the onecutter enzyme releases only one size fragment from the array, the unitsmust be organized in a perfect head-to-tail array.

Derivatives of 3134 cell lines containing pTET-nGFP-C656G andpOP-nGFP-C656G have been generated allowing GFP-GR expression upontetracycline withdrawal from 5 ug/ml or upon induction with IPTG usingstandard procedures and as recommended by the manufacturer of theseinducible systems, respectively. For example, 3616 is a single cellclone of 3134 with the pTET-nGFP-C656G DNA allowing acceptable level ofGFP-GR expression that contains 200 copies of the MMTV LTR-cat-BPVtandem repeats; 3617 is another single cell clone of 3134 with thepTET-nGFP-C656G DNA but where the copy number of the MMTV LTR-cat-BPVtandem repeats dropped from 200 copies to 150 copies upon passage andthen increased to about 170 copies in all cells upon further passage;3596 is a single-cell clone of 134 having 200 copies of the MMTVLTR-cat-BPV repeats and contains pOP-nGFP-C656G DNA; 3597 is a singlecell clone of 3134 with 200 copies of the MMTV LTR-cat-BPV repeats andcontains pOP-nGFP-C656G DNA. Acceptable levels of GFP-GR is reached inthese derivatives after overnight induction of the regulatablepromoters, e.g., after withdrawal of tetracycline in the case of 3616and 3617, and after addition of IPTG in the case of 3596 and 3597.

Gene Targeting with GFP-GR in 3134 Cell

When the GFP-Glucocorticoid Receptor is transfected into the 3 134 cellline, it is initially localized completely in the cytoplasm of the cell,as is normal, non-derivatized glucocorticoid receptor. When GFP-GR isactivated with the GR ligand dexamethasone, the receptor translocates tothe nucleus and accumulates on the BVP/MMTV-LTR/ras tandem array.

FIG. 2 is a schematic representation of the appearance of GFP-GR whenthe fluorescent tag is excited by 489 nm laser light, and the 511 nmemission examined by confocal microscopy, using a standard fluoresceinfilter set. Individual sections are depicted for a representative 3134nucleus. As one focuses on 0.18 micrometer sections through the nucleus,a continuous fiber of intense light emission is detected over 5-7sections, which corresponds to GFP-GR binding to the continuousBVP/MMTV-LTR/ras array.

Expression of GFP-GR in 3134 Cell

1) Materials.

a) Recombinant DNA

The LacSwitch™ inducible mammalian expression system (Stratagene catalognumber 217450) is supplied with the phagemid DNAs: p3'SS forconstitutive expression of the E. coli lac repressor and hygromycinresistance drug selectable marker; pOP13 CAT for lac repressor regulatedexpression from the Rous sarcoma virus (RSV)-LTR promoter and neomycinresistance drug selectable marker; and pOPRSVI CAT for lac repressorregulated expression from the Rous sarcoma virus (RSV)-LTR promoter andneomycin resistance drug selectable marker.

Using standard recombinant DNA methodology, cDNA encoding GFP-GR fusion(either with the C656G mutation in the steroid binding domain of rat GRas present in the plasmid pCI-nGFP-C656G or wild type ligand bindingdomain of rat GR as present in the plasmid pCI-nGFP-rGR) is subclonedinto the vector pOPRSVI CAT phagemid (Stratagene) at the NotI site. ThecDNA should contain at its 5′ end Kozak consensus sequence for efficientutilization of the translational initiation codon ATG, and at its 3′ endthe translational termination sequence followed by multiple translationtermination sequences in all three reading frames. The vector providesRSV-LTR that has been engineered to be regulated by E. coli lacrepressor so that in the mammalian cells with lac repressor, the strongRSV LTR promoter is inducible by the addition of 5 mMisopropyl-beta-D-thiogalactopyranoside (IPTG). The vector also providesan intron in the 5′ untranslated region to ensure proper processing ofthe nascent transcript and maturation into productive mRNA. The vectoralso provides at the 3′ untranslated region, herpes simplex thymidinekinase (TK) polyadenylation signal to ensure polyadenylation and messagestability.

All DNAs used for introduction into mammalian cells are prepared from E.coli by alkali lysis procedure followed by isopycnic centrifugation bybanding twice in cesium chloride/ethidium bromide gradient. Ethidium isremoved by repeated extraction with isopentyl alcohol and cesiumchloride by dialysis against large volumes of 10 mM Tris-HCl, pH 8/1 mMEDTA (TE). After dialysis, DNA is extracted twice in phenol chloroformsolution and then with chloroform before precipitation with 0.2 M sodiumacetate (pH 5.5) and 70% ethanol at −20C. After collecting theprecipitate by centrifugation, the DNA is washed with cold 70% ethanol,air dried, and then resuspended at a concentration of 1-2.5 mg/ml in TE.

b) Cell Line

The 3134 cell line contains a 9 kb repeat of the MMTV LTR fused toHa-v-ras protooncogene in a BPV-based mammalian vector transformed intomouse carcinoma cell line C127.

Derivatives of 3134 cell lines containing pTET-nGFP-C656G andpOP-nGFP-C656G have been generated allowing GFP-GR expression upontetracycline withdrawal from 5 ug/ml or upon induction with IPTG (about1 mM) using standard procedures and as recommended by the manufacturerof these inducible systems. Acceptable levels of GFP-GR is reached inthese derivative cells after overnight induction of the regulatablepromoters.

2) Maintenance of 3134 Cells

The 3134 cells are maintained in complete DMEM media [Dulbecco'sModified Eagle Medium (DMEM; Gibco-BRL catalog number 11965-084)supplement with 2 mM L-glutamine (Gibco-BRL catalog number 25030-024),50 ug/ml gentamicin reagent (Gibco-BRL catalog number 15750-011), and10% fetal bovine serum (Gibco-BRL catalog number 26140-079) as monolayerin 162 cm² cell culture flasks (Costar catalog number 3150) at 37C in 5%CO₂ humidified air incubator. Upon confluence, cells are washed withDulbecco's phosphate buffered saline (D-PBS) without calcium ormagnesium and then treated with 6 ml of 0.05% trypsin/0.53 mM EDTA forseveral minutes at RT (Gibco-BRL catalog number 25300-062). After cellsround up, the side of the flask is tapped to dislodge cells from theflask surface. Repeated pipetting of the cells about three times resultsin a single cell suspension. One fifth of the cell suspension istransferred to a fresh 162 cm² flask containing 25 ml of the completeDMEM media. The freshly diluted cells are returned to the 37C/5% CO₂incubator.

3) Introduction of DNA into 3134 Cells by Electroporation

3134 cells grown to about 90% confluence are harvested using thetrypsin-EDTA and the activity of the trypsin quenched by placing thesingle cell suspension into at least equal volume of complete DMEM.Cells are counted in a hemacytometer and concentrated by centrifugationin bench top centrifuge (Sorvall RT6000D at 2,000 rpm for 5 minutes at4C) after placing in 50 ml sterile conical tube (Falcon 2070).Supernatant is aspirated and cells are washed again using complete DMEM.After the second wash, the resulting cell pellet is resuspended at aconcentration of 20 million cells per 200 ul in cold DMEM and placed onice. In 1.7 ml sterile microtube, appropriate DNA to be transfected,from 1-50 ug of about 8 kb plasmid DNA (typically 1-20 ug), is placed.200 ul 3134 cells is added and mixed well. The mixture is transferred todisposable electro chamber cuvettes (Gibco-BRL catalog number 11601-010)on ice. Cuvettes are placed into electroporation chamber andelectroporated (Gibco-BRL Cell-Porator catalog series 1600; settings:250 volts, 800 or 1180 microFarads, low resistance). Recovery on ice isallowed for 5 minutes. Then, cells are removed from the cuvette andtransferred to 50 ml conical tube with complete DMEM at RT. Cells areplated out on 150×25 mm tissue culture dish (Falcon 3025) in 30 ml ofcomplete DMEM containing about one fourth the content of theelectroporation cuvette. Plates are placed in 37C/5% CO2 incubator. Nextday, media is changed to fresh media and selected with appropriate drug.Every two days media is changed and selection maintained with theappropriate drug.

4) Stable 3134 Cell Line Containing Lac Repressor

Electroporation of 1 ug, 5 ug, 10 ug, 30 ug, and 60 ug p3'SS DNA into 20million 3134 cells in 200 ul cold DMEM is performed as described above.One day following electroporation, cells are selected with 450 ug/mlhygromycin B (Calbiochem catalog number 40005) for about 10 days duringwhich hygromycin resistant colonies arise. The colonies are isolated bytreating with trypsin impregnated sterile 3 mm cloning discs (PGCScientifics catalog number 09-060) and transferred into 24 well tissueculture cluster dish (Costar 3524, each well 16 mm) containing 2 ml ofcomplete DMEM with 450 ug/ml hygromycin B. The cells are allowed to growin the dish in a 37C/5% CO2 incubator until at least 50% confluence.Media is replaced every couple of days.

Cells are trypsinized and transferred to 100×20 mm dish (Falcon 3003)containing 10 ml of complete DMEM with 450 ug/ml hygromycin B. Then, atabout confluence, cell are trypsinized and transferred to 150×25 mm dishwith 30 ml of complete DMEM with 450 ug/ml hygromycin. Near confluence,cells were trypsinized and aliquot replated into 100 mm dish while theremaining cells are frozen down in 10% DMSO with complete DMEM at −70C.Cells grown on 100 mm dishes are harvested at confluence. Extracts areprepared and tested by Western blotting with rabbit anti-lac repressorpolyclonal antibody (Stratagene catalog number 217449) and detectedusing goat anti-rabbit IgG horseradish peroxidase conjugated antibody(Bio-rad catalog number 170-6515) and enhanced chemiluminescent ECLwestern blotting detection reagents (Amersham catalog number RPN 2106).Cells expressing high amounts of the lac repressor are recloned and usedfor electroporating the pOPRSVI CAT plasmid containing GFP-C656G orGFP-rGR fusion cDNA.

5) Stable 3134 Cell Line Containing IPTG-Inducible GFP-GR or GFP-FusionProtein Expression System.

3134 cells showing high levels of constitutive lac repressor expressionwithout cytotoxic effects are used for electroporating pOPRSVI CATplasmid containing the appropriate GFP-fusion protein cDNA. Followingsimilar regiment as described above, the cells are selected with 450ug/ml hygromycin B as well as 500 ug/ml geneticin (Gibco-BRL catalognumber 11811-031). Following about ten days after selection, again drugresistant colonies are isolated, expanded, and characterized.Characterization is achieved by examining uninduced as well as 5 mM IPTGinduced cells using Southern blot analysis of the genomic DNA, Westernblot analysis using antibody directed against GFP and GR or othersteroid receptor, characterize for localization of the GFP-fusionprotein on the MMTV LTR-tandem array upon exposure to the appropriateligand. Cells with appropriate desirable characteristics are subclonedand serve as reagents for diagnostic purposes.

Additional GFP-Steroid Fusion Proteins

The same technology is used to prepare GFP fusions for each member ofthe steroid/thyroid receptor family, including the estrogen receptor,progesterone receptor, androgen receptor, mineralocorticoid receptor,thyroid receptor, retinoic acid receptor (RAR), RXR receptor, vitamin Dreceptor, and TCCD (dioxin) receptor. Chimeric proteins for each of thereceptors is be prepared using GFP fused either to the N-terminus or theC-terminus of the appropriate receptor. The chimeras are tested forfunctional transcriptional transactivation activity, and for binding tocognate response elements using cell lines described below.

GFP-ER: For example, the glucocorticoid receptor portion ofpCI-nGL1-C656G DNA has been replaced with a human estrogen receptor. TheGFP-ER was found to be functional in both transcriptional activation aswell as proper subcellular localization. This further demonstrates theutility of the expression vector as well as the GFP-tagging approach.

Modification of the GFP Fluorescent Moiety

The success of GFP-GR fusion in detecting and characterizing in vivogene targeting indicates that modification and enhancement of the GFPpolypeptide can be of considerable usefulness. These modificationsinclude:

-   1) Changes in the exicitation wavelength to permit activation of    selected GFP fusions;-   2) Changes in the emission wavelength to permit observation of    selected GFP fusions (GFP's will emit with different colors);-   3) Enhancements to the efficiency of excitation and emission (GFP's    will be “brighter”);-   4) Enhancements of GFP-chimeric proteins production by engineering    the GFP sequence for more efficient expression:    -   a) increasing the steady state level of GFP-chimeric protein        mRNA levels through enhanced transcription, transcript        processing, and RNA stability;    -   b) increasing translational efficiency of the GFP-chimeric        proteins by conservative substitutions of the protein coding        regions and/or by introducing codons preferred for translation        in humans cells;    -   c) increasing stability of the GFP-chimeric proteins by        identification of protein destabilizing sequences and selective        elimination of such turnover signals;-   5) Changes in GFP to Define the Minimal Fluorescing Polypeptide that    could Function in a Fusion Protein.

For example, in plasmids pCI-nGL2-C656G; pCI-nGL3-C656G; pCI-nGL4-C656G;pCI-nGL5-C656G; pCI-nGL7-C656G; pCI-nGL9-C656G; pCI-nGL10-C656G;pCI-nGL11-C656G, blue variants of GFP are used. Additionally, in theseplasmids, humanized codons encoding the fusion protein were utilized.

Additional Cell Lines

Human and other mammalian cell lines can be prepared containing receptorbinding sites (receptor response elements) in multimerized arrays fordirect visualization of in vivo gene targeting.

1) For each receptor, these arrays can be created by gene amplification.The receptor response element is fused to the dihydrofolate reductase(dHFR) gene, transfected into human cells, and integrants selected byresistance to methotrexate. After selection of the initial cell lines,further selection is applied using sequentially higher levels ofmethotrexate. This results in amplification of the dHFR/receptorresponse element sequence, providing an array to which the receptor/GFPfusion binding can be observed (directly analogous to the 3134 cell linefor GR). Further detatils are provided below.

2) An alternative approach is to multimerize the receptor responseelement by synthetic DNA synthesis, then introduce this amplifiedelement directly into mammalian cells. This can provide a concentratedtarget for GFP-receptor localization.

Generating Cells having Tandem Arrays

An approach that is being used to generate tandem arrays in are-engineered promoter to obtain a functional transcription unit:

-   1) the MMTV LTR in the plasmid pLTRLuc is being mutated by site    directed mutagenesis to introduce appropriate restriction enzyme    cleavage sites at approximately positions:    +110 from start of transcription (HindIII)    −40 from start of transcription (Sail)    −217 from start of transcription (XhoI)    −1100 from start of transcription (XmaI)-   2) the MMTV LTR in the plasmid pLTRLuc is being mutated by site    directed mutagenesis to introduce appropriate restriction enzyme    cleavage sites at approximately positions:    +110 from start of transcription (HindIII)    −80 from start of transcription (SalI)    −217 from start of transcription (XhoI)    −1100 from start of transcription (Xmal)-   3) the DNA from (1) and (2) are being subcloned into two luciferase    reporter gene vectors from Promega called pGL3-Basic (catalog number    88-1737) and pRL-CMV (catalog number 1068-2003) by using HindIII and    XmaI digests of (1) and (2) and pGL3-Basic DNAs and inserting the    mutated LTR fragments from (1) and (2) into the polylinker region of    pGL3-Basic. In the case of pRL-CMV, this DNA is cleaved with PstI    and BglII and the mutated LTR fragments from (1) and (2) inserted in    place of the CMV Immediate Early Enhancer/Promoter regions in the    pRL-CMV vector using a PstI/HindIII adapter on one end of the    fragment. Also, before the insertion of the mutated LTR DNAs    from (1) and (2), the SalI site present in the pGL3-Basic is    eliminated by digesting this vector with SalI, filling-in the end    with DNA polymerase, and subsequent resealing of the filled-in end    before transforming bacteria.-   4) oligonucleotides containing the glucocorticoid response elements    (GRE: 5′ AGAACAnnnTGTTCT 3′) (SEQ ID NO:4) or estrogen response    elements (ERE: 5′ AGGTCAnnnTGACCT 3′) (SEQ ID NO:5) are synthesized    and then annealed (e.g., oligonucleotide 1 and 2; or oligonucleotide    3 and 4) such that one end contains a cohesive end for SalI and the    other XhoI. Some examples of such oligonucleotides are (where the    lower case letters are spacer bases and in the above designation    would have been designated as “n”):

oligonucleotide 1:5′ tcgagcgcgcaAGAACAcagTGTTCTgacgacacgaAGAACAggaTGTTCTcgtacagtg 3′ (SEQID NO:6): oligonucleotide 2:5′ tcgacactgtacgAGAACAtccTGTTCTtcgtgtcgtcAGAACActgTGTTCTtgcgcgc 3′ (SEQID NO:7): oligonucleotide 3:5′ tcgagcgcgcaAGGTCAcagTGACCTgacgacacgaAGGTCAggaTGACCTcgtacagtg 3′ (SEQID NO:8): oligonucleotide 4:5′ tcgacactgtacgAGGTCAtccTGACCTtcgtgtcgtcAGGTCActgTGACCTtgcgcgc 3′ (SEQID NO:9):

-   5) the annealed oligoncleotides (1 and 2; or 3 and 4) are then    ligated to generate large arrays in a perfect head-to-tail tandem    array; these arrays may go through subcloning steps in bacteria to    build larger arrays from smaller ones as well as to verify the    integrity of the DNA sequence in the array by DNA sequencing.-   6) the multimerized arrays from (5) are then inserted into the    SalI/XhoI site in the mutated LTR of (3).-   7) also, an additional series of constructs are also made lacking    the region from −217 to    −1100 of the LTR by restriction enzyme digestion with Xho I and MluI    for the pGL3-Basic vector derived clones of (6), filling in the    XhoI/MluI cohesive ends, and resealing the blunt ends to generate a    multimerized tandem array of a DNA binding site driving the    expression of the luciferase gene only through the binding sites in    the array and the region of the LTR containing the signal for the    start point of transcription (the TATA box and the initiator    sequence).-   8) stable mammalian cell lines (e.g., from human origin or Chinese    hamster ovary (CHO))are generated with the DNAs from (6) and (7)    after linearization of the DNA, e.g. with BamHI and KpnI for the    pGL3-Basic based DNAs and BamHI for the pRL-CMV based DNAs, and    transfecting or electroporating these DNAs into cells, as is    commonly done to introduce foreign DNA into cell. Also included in    the transfection or electroporation is a DNA for a drug selectable    marker such as neomycin gene or hygromycin that allows for drug    selection (e.g., G418 or hygromycin, respectively) of cells that    have taken up the foreign DNA. In addition, green fluorescent    protein (GFP)-tagged glucocorticoid, estrogen, and orphan receptors    or DNA binding proteins appropriate for the factor DNA-binding DNA    element is also co-transfected or electroporated.-   9) isolated clones from drug selected cells from (8) are    characterized for the number of copies and dispersal of the    introduced DNAs from (6) and (7). Also, characterized is the amount    of fluorescence derived from the GFP tagged-receptors or DNA-binding    protein as well as the functionality of the expressed protein.-   10) because most of the cell lines from (9) will have the foreign    DNA integrated throughout the genome, localization of the GFP-tagged    DNA binding molecule on the arrays in each of these transcription    unit would result in intense fluorescence spot or focus. It is also    possible that an extremely few number of cells will have integrated    the foreign DNA in small arrays which should look like slight    elongated intense spot. The desirable cells are those that contain    visible intense foci due to binding of the GFP-tagged DNA binding    protein on the transcription unit. In the case of the glucocorticoid    receptor, the desirable cell lines are such that agonist    dexamethasone treatment results in accumulation of the GFP-GR on the    transcription unit to generate intense foci but not antagonist RU486    treatment.-   11) DNAs from (6) or (7) will also be ligated to generate tandem    arrays of each transcription unit and cloned into cosmid vector or    phage P1 vector.-   12) the tandem array transcription units will be treated as in (8).-   13) the result of (11) will be characterized as in (9) and (10) to    obtain cell lines with desirable properties.-   14) DNA from (6) and (7) will be linked to a constitutively    expressible dihydrofolate reductase (DHFR) gene by standard DNA    subcloning techniques.-   15) the DNA from (14) will be introduced into mammalian cells    lacking the tumor suppresor gene, p53, using a similar procedure as    in (8) except leaving out the drug selectable marker DNAs (due to    the fact that DHFR gene is a drug selectable marker gene) and then    selected for two weeks with methotrexate at a concentration about    four times above the LD90. Selection media is replaced every 2-3    days. After methotrexate resistant colonies appear (in approximately    two weeks), the colonies are pooled and part of the pool is frozen    down while the remainder is used in a repeat selection with a    concentration of methotrexate about four times higher than that    previously used. This last selection procedure is repeated a number    of times to finally select out cells with a highly amplified tandem    array copies of DNAs containing the multimerized binding sites, the    reporter gene, and the DHFR gene.-   16) the cell lines after the last methotrexate selection are    characterized for the presence of at least one tandem array and    localization of the GFP-tagged DNA binding protein on this array    analyzed. Localization on this array is manifested as a long linear    highly fluorescent structure observed in the nucleus. In the case of    GFP-GR, the localization on the array should occur upon treatment    with agonist dexamethasone but not antagonist RU486.-   17) other approaches to generating array envisioned is the use of    site specific recombinases in vivo to generate precisely tailored    tandem arrays.-   18) while items 1-16 focuses on the use of a genetically engineered    artificial promoter/enhancer to generate arrays, tandem arrays may    be made from just binding sites only or from natural regulatory DNAs    harboring desirable factor-DNA binding sites.-   19) since the higher eukaryotic genomes contain naturally occuring    repetitive sequences, interactions of appropriately tagged    DNA-binding protein with such naturally occurring sequences may also    prove useful.

Using this general approach, functional receptor/cell line pairs can beestablished for each receptor-GFP fusion. These reagents provide asimple, rapid, straightforward, sensitive, and biologically relevantassay for each receptor.

Using standard recombinant DNA methodology, DNA encoding GFP-fusionprotein sequences can be placed under the control of high expressioneukaryotic promoter/enhancers (e.g. CMV promoter/enhancer, SV40promoter/enhancer, RSV LTR,herpes simplex thymidine kinase [TK]promoter, etc.), naturally occurring inducible promoter/enhancers (e.g.metallothionine promoter/enhancer, MMTV LTR, heatshockpromoter/enhancer, etc.), or synthetic inducible promoter/enhancers(e.g. GAL4-VP16 inducible system, Stratagene's LacSwitch™ induciblemammalian expression system (catalog number 217450), Life Technologies'Tet regulated expression system (catalog number 10583-011), etc.). TheseDNAs are introduced into mammalian cells along with DNA expressing aselectable marker (neomycin, hygromycin, zeocin, etc.) or screenablemarker (e.g. fluorescence, foci formation, etc.) by standard protocol(e.g.calcium phosphate co-precipitation, electroporation,liposome-mediated transfection, viral infection, etc.). Followingrecovery of the cells to the introduction of DNA, approximately 1-3 dayslater, selectable agent is applied in the case of drug selectable markerto select for cells with stable integration of the selectable marker.

Upon continuous culturing of the cells under selection condition for anadditional week during which clonal populations of cells will arise, theresulting cells are then characterized for the presence of the stablyintegrated DNA by Southern blot analysis and PCR analysis, for theexpression of the GFP-fusion protein by Western blot analysis, flowcytometry, and microscopic examination, for participation inligand-dependent translocation/transactivation by assaying the activityof reporter genes (e.g.chloramphenicol acetyltransferase, luciferase,beta-galactosidase, etc.) under the control of the appropriate ligandinducible promoter/enhancer (e.g., glucocorticoid response elementcontaining promoter, estrogen response element containing promoter,etc.), and for localization on the target sequences in nuclei ofexpressing cells by high resolution fluorescence imaging systems (e.g.confocal laser scanning microscopy, cooled CCD camera microscopy, etc.).Alternatively, stable transformants expressing GFP-fusion proteins canbe directly isolated by fluorescence activated cell sorting (FACS) usingappropriate excitation wavelengths and emission detector.

Although less preferable at present, an alternative to isolating clonesof stable transformants will be the isolation of pools of stabletransformants. The use of cell line/expression system combination maynecessitate such a generation of a pool of stable transformants.

Additionally, while it is presently most efficient to obtain stable celllines, GFP-fusion expression plasmid can be transiently introduced intocells and analyzed for ligand-dependent translocation/nuclear targeting.

Use of Reagents

Because the use of GFP as a tag involves fusing a rather large protein(27 kDa) to GR, it was examined whether any GR activity has beencompromised by GFP. To this end, GFP was fused to a rat GR with theC656G point mutation. This allowed selectively activation of GFP-GRwithout activating the endogenous receptor, and, thus, to assess theactivity of the chimeric receptor independently of the endogenous GR.From the dose response curve and transcriptional activation of the MMTVLTR reporter gene (FIG. 3B), it is clear that GFP-GR can be selectivelyactivated without activating the endogenous receptor.

By a number of criteria, GFP-GR functions very much like GR. Inparticular, the tagged receptor resides in the cytoplasm until activatedby a ligand; it then translocates into the nucleus at a rate comparableto that previously reported (22). The rate and extent of GFP-GRtranslocation shows a dependence on the concentration of the activatingligand as well as a ligand specificity reflective of the nativereceptor. Furthermore, since both dexamethasone and RU486 treatment leadto complete translocation of GFP-GR from the cyoplasm to the nucleus inall cells, essentially all of the GFP-GR molecules exist in aconformation competent for both ligand binding and nucleartranslocation. Once in the nucleus, GFP-GR's ability to activate thetranscription of a MMTV LTR reporter gene depends on the type ofactivating ligand, consistent with previous results for GR. In the caseof a potent agonist, dexamethasone, less GFP-GR is required foractivation of transiently introduced MMTV LTRLuc reporter gene than forthe endogenous GR, indicating that even with respect to transactivationpotential, the presence of GFP has not altered the transcriptionalpotency ascribed to the C656G point mutation. Thus, in all aspects, thepresence of GFP appears not to have affected normal GR function and hasa general utility for studying the mechanisms of transcriptionalregulation in vivo.

Since the S65T variant of GFP used here is highly excitable at 499 nmwavelength and resistant to photobleaching, it was possible to followthe course of cytoplasm-to-nuclear translocation of GR in a singleliving cell for an extended period of time. Upon binding todexamethasone, GFP-GR moves vectorially toward the nucleus. Inhibitionof import by chilling indicates that this transport is facilitated.Accumulation of GFP-GR along fibrillar structures before dexamethasoneaddition, and in the perinuclear region after dexamethasone addition,suggests that the cytoskeleton is involved in the transport process.Finally, the pulsatile brightness changes in the perinuclear regionsupport an energy- and microtubule-dependent active transport process.

The rate of translocation of GFP-GR was dependent on hormoneconcentration, reflecting the dose- and time-dependence of GR action.This suggests that the rate of translocation contributes to GR function.The present invention indicates that cells having multiple copies of theresponse element in fluorescently detectable array can be a useful modelto study reagents that modify rates of nuclear translocation andresponse element binding.

Finally, it is well-known that DNA in the nucleus is localized in anon-random fashion. The study of the pattern of nuclear fluorescencewith GFP-GR supports the idea of an inherent order in the organizationof the interphase nuclei and may reflect structures related to thisorganization. The reproducibility in the pattern of GFP-GR accumulationbetween neighboring nuclei reflects an inherent order of the interphasenucleus with regards to both GR-target site architecture (the foci ofbright fluorescence) as well as transcriptionally incompetent GFP-GR,which appears to accumulate in a reticular pattern, reminiscent ofassociation with the nuclear matrix. Thus, functional differences inGFP-GR due to hormone-specific effects (e.g., dexamethasone vs. RU486)are reflected in the patterns of GFP-GR intranuclear accumulation,indicating a general utility of understanding intranuclear localizationof GR in addressing hormone-mediated actions.

Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompaying claims.

What is claimed is:
 1. A method of screening for a ligand that activatesthe translocation of a steroid receptor to the nucleus in a mammaliancell comprising: a. contacting a mammalian cell having a nucleus withthe ligand, wherein the cell has a plurality of steroid receptorresponse elements, wherein the steroid receptor response elementscomprise a plurality of AGAACA (SEQ ID NO:4) or AGGTCA (SEQ ID NO:5), inan array such that the response element can be directly detected whenbound by fluorescently labeled steroid receptor; and b. detecting thelocation of fluorescence within the cell, a change in the relativefluorescence of the nucleus to the cytoplasm so as to increase thefluorescence of the nucleus indicating a ligand that activates thetranslocation of a steroid receptor to the nucleus in a mammalian cell.2. The method of claim 1, wherein the fluorescently labeled steroidreceptor is fluorescently labeled with a green fluorescent protein.
 3. Amethod of screening for a ligand that activates the translocation of asteroid receptor to the nucleus in a mammalian cell comprising: a.contacting a mammalian cell having a nucleus with the ligand, whereinthe cell has a plurality of steroid recentor response elements, whereinthe steroid receptor response elements comprise a plurality of AGAACA(SEQ NO:4) or AGGTCA (SEQ ID NO:5), in an array such that the responseelement can be directly detected when bound by fluorescently labeledsteroid receptor, and b. detecting the location of fluorescence withinthe cell, a change in the relative fluorescence of the nucleus to thecytoplasm so as to increase the fluorescence of the nucleus indicating aligand that activates the translocation of a steroid receptor to thenucleus in a mammalian cell, wherein the mammalian cell is a cell of thecell line designated 3134 deposited with American Type CultureCollection under accession number CRL-11998 (ATCC).